Methods and compositions for treating aging-associated impairments

ABSTRACT

Methods of treating an adult mammal for an aging-associated impairment are provided. Aspects of the methods include reducing the β2-microglobulin (B2M) level in the mammal in a manner sufficient to treat the mammal for the aging-associated impairment. A variety of aging-associated impairments may be treated by practice of the methods, which impairments include cognitive impairments.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/574,795, filed Nov. 16, 2017, which is a U.S. 371 national phaseentry of International Patent Application No. PCT/US2016/032907, filedMay 17, 2016, which claims priority to the filing date of the U.S.Provisional Patent Application Ser. No. 62/163,222 filed May 18, 2015,the disclosure of which applications are incorporated herein byreference in their entireties.

This application is also a continuation-in-part application of U.S.patent application Ser. No. 14/280,939 filed on May 19, 2014; whichapplication is a continuation application of U.S. patent applicationSer. No. 13/575,437 filed on Oct. 9, 2012, now abandoned; whichapplication is a United States national phase application of PCTApplication Serial No. PCT/US2011/022916 filed on Jan. 28, 2011; whichapplication, pursuant to 35 U.S.C. § 119 (e), claims priority to thefiling date of the U.S. Provisional Patent Application Ser. No.61/298,998 filed Jan. 28, 2010; the disclosures of which applicationsare incorporated herein by reference.

GOVERNMENT RIGHTS

This invention was made with Government support under contractsAG027505, OD012178, and TR000004 awarded by the National Institutes ofHealth. The Government has certain rights in the invention.

INTRODUCTION

Aging in an organism is accompanied by an accumulation of changes overtime. In the nervous system, aging is accompanied by structural andneurophysiological changes that drive cognitive decline andsusceptibility to degenerative disorders in healthy individuals. (Heeden& Gabrieli, “Insights into the ageing mind: a view from cognitiveneuroscience,” Nat. Rev. Neurosci. (2004) 5: 87-96; Raz et al.,“Neuroanatomical correlates of cognitive aging: evidence from structuralmagnetic resonance imaging,” Neuropsychology (1998) 12:95-114; Mattson &Magnus, “Ageing and neuronal vulnerability,” Nat. Rev. Neurosci. (2006)7: 278-294: and Rapp & Heindel, “Memory systems in normal andpathological aging,” Curr. Opin. Neurol. (1994) 7:294-298). Included inthese changes are synapse loss and the loss of neuronal function thatresults. Thus, although significant neuronal death is typically notobserved during the natural aging process, neurons in the aging brainare vulnerable to sub-lethal age-related alterations in structure,synaptic integrity, and molecular processing at the synapse, all ofwhich impair cognitive function.

In addition to the normal synapse loss during natural aging, synapseloss is an early pathological event common to many neurodegenerativeconditions, and is the best correlate to the neuronal and cognitiveimpairment associated with these conditions. Indeed, aging remains thesingle most dominant risk factor for dementia-related neurodegenerativediseases such as Alzheimer's disease (AD) (Bishop et al., “Neuralmechanisms of ageing and cognitive decline,” Nature (2010) 464: 529-535(2010); Heeden & Gabrieli, “Insights into the ageing mind: a view fromcognitive neuroscience,” Nat. Rev. Neurosci. (2004) 5:87-96; Mattson &Magnus, “Ageing and neuronal vulnerability,” Nat. Rev. Neurosci. (2006)7:278-294).

As human lifespan increases, a greater fraction of the populationsuffers from aging-associated cognitive impairments, making it crucialto elucidate means by which to maintain cognitive integrity byprotecting against, or even counteracting, the effects of aging (Hebertet al., “Alzheimer disease in the US population: prevalence estimatesusing the 2000 census,” Arch. Neurol. (2003) 60:1119-1122; Bishop etal., “Neural mechanisms of ageing and cognitive decline,” Nature (2010)464:529-535).

β-2 microglobulin (B2M) is a component of the class I majorhistocompatibility complex (MHC), a multi-protein complex found on thesurface of nearly all nucleated mammalian cells. These complexesfunction by presenting foreign antigens or peptide fragments on the cellsurface so that the immune system may recognize and destroy infectedcells. The protein components of the class I MHC are encoded by severalgenes, each with multiple alleles, and the types of expressed class IMHC's vary among individuals. Because the MHC is polymorphic, it is animportant factor for consideration during organ transplant as the hostimmune system may reject organs with foreign MHC's. In cancerous cells,MHC expression may be defective, allowing such cells to escape immunedetection and destruction.

Free extracellular B2M is also found in human physiological fluids suchas the blood serum, urine, and cerebral spinal fluid. Due to its smallsize, the protein is normally filtered from the blood and thenreabsorbed in some amount by the kidney. High serum concentrations ofB2M often accompany the presence of several diseases such as non-Hodgkinlymphoma and meningitis (Hallgren et al., “Lactoferrin, lysozyme, andbeta 2-microglobulin levels in cerebrospinal fluid: differential indicesof CNS inflammation,” Inflammation (1982) 6:291-304; et al., “Prognosticsignificance of serum beta-2 microglobulin in patients with non-Hodgkinlymphoma,” Oncology (2014) 87:40-7). When present in body serum at highconcentrations, the protein can form amyloid fibrils (Corland &Heegaard, “B (2)-microglobulin amyloidosis,” Sub-cellular Biochemistry(2012) 65:517-40). The buildup of B2M in body tissue and fluids as acomplication of chronic kidney disease in individuals on dialysis hasbeen extensively studied. In patients with reduced kidney function,buildup is associated with joint and bone weakness and pain. Urine B2Mlevels are measured to indicate kidney damage and filtration disorders(Acchiardo et al., “Beta 2-microglobulin levels in patients with renalinsufficiency,” American Journal of Kidney Diseases (1989) 13:70-4;Astor et al., “Serum Beta-2-microglobulin at discharge predictsmortality and graft loss following kidney transplantation,” KidneyInternational (2013) 84:810-817).

Because protein aggregates of B2M play a role in provokingosteoarthritis, there is concern that the protein may be toxic toneuronal cells sensitive to abnormal protein deposits (Giorgetti et al.,“beta2-Microglobulin is potentially neurotoxic, but the blood brainbarrier is likely to protect the brain from its toxicity.” NephrologyDialysis Transplantation (2009) 24:1176-81). The protein has beenimplicated in neuronal development, normal hippocampus dependent memoryand synapse formation and plasticity (Bilousova et al., “Majorhistocompatibility complex class I molecules modulate embryonicneuritogenesis and neuronal polarization,” Journal of Neuroimmunology(2012) 247:1-8; Harrison et al., “Human brain weight is correlated withexpression of the ‘housekeeping genes’ beta-2-microglobulin andTATA-binding protein,” Neuropathology and Applied Neurobiology (2010)36:498-504). Changes in proteins of the class I MHC such as beta 2microglobulin could disrupt synaptic plasticity and lead to cognitivedeficits in an aging, damaged, or diseased brain (Nelson at al., “MHCclass I immune proteins are critical for hippocampus-dependent memoryand gate NMDAR-dependent hippocampal long-term depression,” Learning &Memory (2013) 20:505-17). A deficiency in B2M may also result in theloss of left-right asymmetries in the hippocampal region of the brain(Kawahara et al., “Neuronal major histocompatibility complex class Imolecules are implicated in the generation of asymmetries in hippocampalcircuitry,” The Journal of Physiology (2013) 591:4777-91).

In addition, B2M serves as a molecular marker that can be used todetermine immune compromise or central nervous system immune activation(Svatonfova et al., “Beta2-microglobulin as a diagnostic marker incerebrospinal fluid: a follow-up study,” Disease Markers (2014) 2014).Levels of the protein may signify the extent of the central nervoussystem inflammatory response. A review of B2M and its use as a diseasemarker states that elevated levels of B2M in the cerebral spinal fluidis reflective of multiple sclerosis, neuro-Behçet's disease,sarcoidosis, acquired immunodeficiency syndrome-dementia complex andmeningeal metastasis of malignant tumors (Adachi, “Beta-2-microglobulinlevels in the cerebrospinal fluid: their value as a disease marker. Areview of the recent literature,” European Neurology (1991) 31:181-5).Other studies suggest that B2M could potentially serve as a clinicalmarker for cognitive impairment risk or a tool for disease prognosis forindividuals experiencing a range of diseases including kidney failure,HIV infection, and Alzheimer's (Almeida, “Cognitive impairment and majordepressive disorder in HIV infection and cerebrospinal fluidbiomarkers,” Arquivos de Neuro-Psiquiatria (2013) 71:689-92; Annunziataat al., “Serum beta-2-microglobulin levels and cognitive function inchronic dialysis patients,” Clinica Chimica Acta (1991) 201:139-41;Doecke et al., “Blood-based protein biomarkers for diagnosis ofAlzheimer disease,” Archives of Neurology (2012) 69:1318-25; Isshiki etal., “Cerebral blood flow in patients with peritoneal dialysis by aneasy Z-score imaging system for brain perfusion single photon emissiontomography,” Therapeutic Apheresis and Dialysis (2014) 18:291-6).Elevated serum levels hold particular prognostic significance for adultmultiple myeloma, lymphocytic leukemia and lymphoma (Kantarjian at al.,“Prognostic significance of elevated serum beta 2-microglobulin levelsin adult acute lymphocytic leukemia,” The American Journal of Medicine(1992) 93:599-604; Wu et al., “Prognostic significance of serum beta-2microglobulin in patients with non-Hodgkin lymphoma,” Oncology (2014)87:40-7). More studies continue to explore the implications of abnormalserum and tissue B2M levels for cancer, cardiovascular disease,schizophrenia, and systemic disease activity (Chittiprol et al.,“Longitudinal study of beta2-microglobulin abnormalities inschizophrenia,” International Immunopharmacology (2009) 9:1215-7). Insome cases, B2M has been the target of disease therapies (Morabito etal., “Analysis and clinical relevance of human leukocyte antigen classI, heavy chain, and beta2-microglobulin down regulation in breastcancer,” Human Immunology (2009) 70:492-5; Yang et al., “Identificationof beta2-microglobulin as a potential target for ovarian cancer,” CancerBiology & Therapy (2009) 8:232-8).

SUMMARY

Methods of treating an adult mammal for an aging-associated impairmentare provided. Aspects of the methods include reducing theβ2-microglobulin (B2M) level in the mammal in a manner sufficient totreat the mammal for the aging-associated impairment. A variety ofaging-associated impairments may be treated by practice of the methods,which impairments include cognitive impairments.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1a-1k . B2M are a component of the aging systemic environment thatimpairs hippocampal-dependent cognitive function and adult neurogenesis.FIGS. 1a & 1 c, Schematic of unpaired young versus aged mice (FIG. 1a ),and young isochronic versus heterochronic parabionts (FIG. 1c ). FIGS.1b & 1 c, Changes in plasma concentration of B2M with age at 3, 6, 12,18 and 24 months (FIG. 1b ) and between young isochronic and youngheterochronic parabionts five weeks after parabiosis (FIG. 1d ). Datafrom 5 mice per group. FIGS. 1e & 1 f, Changes in plasma (FIG. 1e ;r=0.51; p<0.0001; 95% confidence interval=0.19-0.028) and CSF (FIG. 1f )B2M concentrations with age in healthy human subjects. FIGS. 1g & 1 k,Young adult (3 months) mice were injected intraorbitally with B2M or PBS(vehicle) control five times over 12 days. FIG. 1g , Schematic ofillustrating the chronological order used for B2M treatment andcognitive testing. FIGS. 1h & 1 i, Hippocampal learning and memory wasassessed by RAWM (FIG. 1h ) and contextual fear conditioning (FIG. 1i ).FIG. 1h , Number of entry arm errors prior to finding platform. i,Percent freezing time 24 h after training. Data from 9-10 mice pergroup. FIG. 1j , Representative field of Dcx-positive cells for eachtreatment group (scale bar 100 μm). FIG. 1k , Quantification ofneurogenesis in the dentate gyrus (DG) after treatment. Data from 7-8mice per group. All data represented as dot plots with Mean or bargraphs with Mean±SEM; *P<0.05; **P<0.01; **P<0.001 t-test (FIGS. 1d, 1f,1i & 1 k), ANOVA, Tukey's post-hoc test (FIG. 1b ), Mann-Whitney U Test(e) and repeated measures ANOVA, Bonferroni post-hoc test (FIG. 1k ).

FIGS. 2a-2e . Hippocampal dependent learning and memory. FIGS. 2a-2e ,Learning and memory was examined during normal aging in young(3-month-old) versus old (18-month-old) animals using RAWM (FIGS. 2a & 2b) and contextual fear conditioning (FIGS. 2c & 2 e) paradigms. n=10 pergroup. FIG. 2a , Old mice demonstrate impaired learning and memory forplatform location during the testing phase of the RAWM task. Cognitivedeficits were quantified as the number of entry arm errors made prior tofinding the target platform. FIG. 2b , No differences in swim speeds ofwere detected between young and old animals. FIG. 2c , Young and oldanimals exhibited similar baseline freezing time during fearconditioning training. FIG. 2d , During contextual fear conditioning oldmice demonstrate decreased freezing time during contextual memorytesting. FIG. 2e , No differences in cued memory were detected 24 hoursafter training. Data represented as mean±s.e.m.; *P<0.05; *P<0.01; n.s.,not significant; t-test (FIGS. 2a-2c & 2 e), repeated measures ANOVA,Bonferroni post-hoc test (FIG. 2d ).

FIGS. 3a-3d . Weight, swim speeds and cued memory are not altered bysystemic B2M administration. FIGS. 3a-3d . Young adult (3 months) micewere injected intraorbitally with B2M or PBS (vehicle) control fivetimes over 10 days prior to behavioral testing. FIG. 3a , Average mouseweight of B2M and vehicle treated groups. FIG. 3b , Swim speeds of miceinjected with B2M or vehicle during the testing phase of the RAWM. FIGS.3c & 3 d, Conditioned fear was displayed as freezing behavior. FIG. 3c ,Animals from all treatment groups exhibited similar baseline freezingtime during training. FIG. 3d , No differences in cued memory weredetected between groups when re-exposed to the conditioned stimulus(tone and light) in a novel context 24 hours after training. Data from 9mice per group. All data represented as Mean+SEM; n.s. not significant;t-test.

FIGS. 4a & 4 b. Systemic administration of B2M decreases neurogenesis inthe DG of young animals. FIGS. 4a & 4 b, Young adult mice (3-4 months)were injected with B2M or PBS (vehicle) control through intraorbitalinjections five times over 12 days. Prior to euthanasia Bromodeoxyuridne(BrdU) was administered by intraperitoneal injections for three days.Quantification of MCM2-positive and BrdU-positive in the dentate gyrus(DG) after treatment. Data from 5 mice per group. All data representedas Mean+SEM; *P<0.05; **P<0.01; t-test.

FIGS. 5a-5h . Local B2M expression increases in the hippocampus duringaging and impairs hippocampal-dependent cognitive function and adultneurogenesis. FIGS. 5a & 5 b, Representative Western blot andquantification of hippocampal lysates probed with anti-B2M andanti-Actin antibodies from young (3 months) and old (18 months) unpairedanimals (FIG. 5a ), or young isochronic and young heterochronicparabionts five weeks after parabiosis (FIG. 5b ). FIGS. 5c-5e , Youngadult (3 months) wild type (WT) and transporter associated with antigenprocessing 1 knock out (Tap1−/−) mice were given unilateral stereotaxicinjections of B2M or vehicle control FIG. 5c , Representative field ofDcx-positive cells in adjacent sides of the DG within the same sectionare shown for WT and Tap1−/− treatment groups. FIGS. 5d & 5 e,Quantification of neurogenesis in the DG of WT (d) and Tap1−/−(FIG. 5e )mice after stereotaxic B2M administration. Data from five mice pergroup.

FIGS. 5f-5h , Young adult mice were given bilateral stereotaxicinjections of B2M or vehicle six days prior to behavioral testing. FIG.5f , Schematic illustrating chronological order used for local B2Madministration and cognitive testing. FIGS. 5g & 5 h, Learning andmemory was assessed by RAWM (FIG. 5h ) and contextual fear conditioning(FIG. 5g ) following stereotaxic injections. Data from 10 animals pergroup. All data represented as Mean±SEM; *P<0.05; **P<0.01; n.s. notsignificant; ANOVA, t-test (FIGS. 5a,5b,5d,5e & 5 h); repeated measuresANOVA, Bonferroni post-hoc test (FIG. 5g ).

FIGS. 6a-6c . Swim speeds and cued memory are not altered by local B2Madministration. FIGS. 6a-6c , Young adult mice were given bilateralstereotaxic injections of B2M or PBS (vehicle) control six days prior tobehavioral testing. FIG. 6a , Swim speeds of mice injected with B2M orvehicle during the testing phase of the RAWM. FIG. 6b , Animals from alltreatment groups exhibited similar baseline freezing time during fearconditioning training. FIG. 6c , No differences in cued memory weredetected between groups when re-exposed to the conditioned stimulus(tone and light) in a novel context 24 hours after training. Data from10 mice per group. All data represented as Mean+SEM; n.s. notsignificant; t-test.

FIGS. 7a-7e . No differences in neurogenesis are observed in the DG ofyoung unpaired or young isochronic WT and Tap1−/− animals. FIG. 7a ,Quantification of Doublecortin (Dcx)-positive cells in the DG of youngadult (3 months) wild type (WT) and Tap1−/− unpaired mice. Data from 5mice per group. FIG. 7b , Schematic of young WT and Tap1−/− isochronicparabionts. FIGS. 7c-7e , Quantification of Dcx, T-box transcriptionfactor Tbr2, and BrdU immunostaining of young WT and Tap1−/− isochronicparabionts five weeks after parabiosis. Data from 6-8 mice per group.All data represented as Mean+SEM; n.s. not significant; t-test (FIG. 7a); ANOVA, Tukey's post-hoc test (FIGS. 7c-7e ).

FIGS. 8a-8d . Reducing endogenous MHC I surface expression mitigates inpart the negative effects of heterochronic parabiosis on adultneurogenesis in young animals. FIG. 8a , Schematic of young wild type(WT) and Tap1 knock out (Tap1−/−) isochronic parabionts and young WT andTap1−/− heterochronic parabionts. FIGS. 8b & 8 c Representative fields(FIG. 8b ) and quantification (FIG. 8c ) of Doublecortin immunostainingof young isochronic and heterochronic parabionts five weeks afterparabiosis (arrowheads point to individual cells, scale bar: 100 μm).FIG. 8d , Prior to euthanasia animals were injected withBromodeoxyuridne (BrdU) for three days, and proliferating cells havingincorporated BrdU were quantified in DG after parabiosis. Data from 8young isochronic WT, 6 young isochronic Tap1−/−, 8 young heterochronicWT, and 8 young heterochronic Tap1−/− parabionts. All data representedas Mean±SEM; *P<0.05; ANOVA, Tukey's post-hoc test.

FIGS. 9a & 9 b. Reducing endogenous MHC I surface expression mitigatesin part the decrease in neuronal progenitor cell number in young miceafter heterochronic parabiosis. FIG. 9a , Schematic of young wild type(WT) and Tap1 knock out (Tap1−/−) isochronic parabionts and young WT andTap1−/− heterochronic parabionts. FIG. 9b , Quantification of the T-boxtranscription factor Tbr2 immunostaining of young isochronic andheterochronic parabionts five weeks after parabiosis Data from 8 youngisochronic WT, 6 young isochronic Tap1−/−, 8 young heterochronic WT, and8 young heterochronic Tap1−/− parabionts. All data represented asMean±SEM; *P<0.05; ANOVA. Tukey's post-hoc test.

FIGS. 10a-10j . Absence of endogenous B2M enhances hippocampal-dependentcognitive function and adult neurogenesis in old animals. FIGS. 10a-10d, Learning and memory was assessed in young (3 months) and old (15-16months) wild type (WT) and B2M knock out (B2M−/−) mice by RAWM (FIGS.10a, 10c ) and contextual fear conditioning (FIGS. 10b & 10 d). Datafrom 10 young and 8-12 old mice per genotype. FIGS. 10e-10j ,Neurogenesis was analyzed by immunostaining for Dcx-positive cells inthe DG of young and old WT and B2M−/− mice. Representative field andquantification of Dcx-positive cells are shown for young (FIGS. 10e & 10f) and old (FIGS. 10e & 10 g) WT and B2M−/− animals (arrowheads point toindividual immature neurons, scale bar: 100 m). Data from 8 young and 10old mice per genotype. FIGS. 10h & 10 j, WT and B2M−/− mice wereadministered BrdU by intraperitoneal injections for six days andeuthanized 28 days later. FIG. 10h , Representative confocal microscopyfrom the DG of brain sections immunostained for BrdU (red) incombination with NeuN (green). FIGS. 10i & 10 j, Quantification of therelative number of BrdU and NeuN-double positive cells out of the totalBrdU-positive cells in the young (FIG. 10i ) and old (FIG. 10j ) DG ofWT and B2M−/− animals. Data from 8 mice per group (3 sections permouse). All data represented as Mean±SEM; *P<0.05; **P<0.01; n.s. notsignificant; t-test (FIGS. 10b, 10d, 10f, 10i, 10j ); repeated measuresANOVA, Bonferroni post-hoc test (FIG. 10a , FIG. 10c ).

FIGS. 11a-11f . Swim speeds and cued memory are not altered in oldB2M−/− animals. FIGS. 11a-11f , Hippocampal learning and memory wasassessed old adult (17 months) WT and during the testing phase of theRAWM. Animals exhibited similar baseline freezing time during fearconditioning training regardless of genotype. No differences in cuedmemory were detected between genotypes when mice were re-exposed to theconditioned stimulus (tone and light) in a novel context 24 hours aftertraining. Data from 12 WT and 8 B2M−/− mice. All data represented asMean+SEM; n.s. not significant; t-test.

FIGS. 12a-12e . Absence of endogenous B2M increases proliferation butnot astrocyte differentiation in an age-dependent manner in vivo. FIGS.12a-12c , To assess proliferation young (3 months) and old (15-16months) wild type (WT) and B2M knock out (B2M−/−) mice were administeredBrdU by intraperitoneal injections for three days prior to euthanasia.FIGS. 12b & 12 c, Immunostaining of BrdU-positive cells was quantifiedin the DG of young (FIG. 12b ) and old (FIG. 12c ) animals. Data from 8young and 10 old mice per genotype. FIGS. 12c-12e , For examineastrocyte differentiation WT and B2M−/− mice were administered BrdU byintraperitoneal injections for six days and euthanized 28 days later.FIG. 12c , Representative confocal microscopy from the DG of brainsections immunostained for BrdU (red) in combination with GFAP (blue).FIGS. 12d & 12 e, Quantification of the relative number of BrdU andGFAP-double positive cells out of the total BrdU-positive cells in theyoung (FIG. 12d ) and old (FIG. 12e ) DG of WT and B2M−/− animals. Datafrom 8 mice per group (3 sections per mouse). All data represented asMean+SEM; **P<0.01; n.s. not significant; t-test.

FIG. 13. Relative levels of beta2-microglobulin were determined inplasma samples of healthy male human donors of 18, 30, 45, 55, and 66years of age by the SomaScan Proteomic Assay (Somalogic, Inc, Boulder,Colo.). For each age group, plasma from 40 individuals was analyzed as 8pools of 5 individuals per pool. Statistical analysis was performed bytwo-sided Student's t-test of log-transformed values, and also bytrend-analysis of untransformed data using the Jonckheere-Terpstra test.Observed changes were found to be highly significant with the p-value ofthe t-test being 1.1×10⁻⁴ (66 vs 18 year old) and the p-value for theJT-test being 1.3×10⁻⁷ (all age groups). (RFU refers to “relativefluorescence units” by SomaScan Proteomic Assay.)

DETAILED DESCRIPTION

Methods of treating an adult mammal for an aging-associated impairmentare provided. Aspects of the methods include reducing theβ2-microglobulin (B2M) level in the mammal in a manner sufficient totreat the mammal for the aging-associated impairment. A variety ofaging-associated impairments may be treated by practice of the methods,which impairments include cognitive impairments.

Before the present methods and compositions are described, it is to beunderstood that this invention is not limited to a particular method orcomposition described, as such may, of course, vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting, since the scope of the present invention will be limited onlyby the appended claims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimits of that range is also specifically disclosed. Each smaller rangebetween any stated value or intervening value in a stated range and anyother stated or intervening value in that stated range is encompassedwithin the invention. The upper and lower limits of these smaller rangesmay independently be included or excluded in the range, and each rangewhere either, neither or both limits are included in the smaller rangesis also encompassed within the invention, subject to any specificallyexcluded limit in the stated range. Where the stated range includes oneor both of the limits, ranges excluding either or both of those includedlimits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, some potential andpreferred methods and materials are now described. All publicationsmentioned herein are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited. It is understood that the present disclosuresupersedes any disclosure of an incorporated publication to the extentthere is a contradiction.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which may be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentinvention. Any recited method can be carried out in the order of eventsrecited or in any other order which is logically possible.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “acell” includes a plurality of such cells and reference to “the peptide”includes reference to one or more peptides and equivalents thereof,e.g., polypeptides, known to those skilled in the art, and so forth.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

Methods

As summarized above, aspects of the invention include methods oftreating an aging-associated impairment in an adult mammal. Theaging-associated impairment may manifest in a number of different ways,e.g., as aging-associated cognitive impairment and/or physiologicalimpairment, e.g., in the form of damage to central or peripheral organsof the body, such as but not limited to: cell injury, tissue damage,organ dysfunction, aging-associated lifespan shortening andcarcinogenesis, where specific organs and tissues of interest include,but are not limited to skin, neuron, muscle, pancreas, brain, kidney,lung, stomach, intestine, spleen, heart, adipose tissue, testes, ovary,uterus, liver and bone; in the form of decreased neurogenesis, etc.

In some embodiments, the aging-associated impairment is anaging-associated impairment in cognitive ability in an individual, i.e.,an aging-associated cognitive impairment. By cognitive ability, or“cognition”, it is meant the mental processes that include attention andconcentration, learning complex tasks and concepts, memory (acquiring,retaining, and retrieving new information in the short and/or longterm), information processing (dealing with information gathered by thefive senses), visuospatial function (visual perception, depthperception, using mental imagery, copying drawings, constructing objectsor shapes), producing and understanding language, verbal fluency(word-finding), solving problems, making decisions, and executivefunctions (planning and prioritizing). By “cognitive decline”, it ismeant a progressive decrease in one or more of these abilities, e.g., adecline in memory, language, thinking, judgment, etc. By “an impairmentin cognitive ability” and “cognitive impairment”, it is meant areduction in cognitive ability relative to a healthy individual, e.g.,an age-matched healthy individual, or relative to the ability of theindividual at an earlier point in time, e.g., 2 weeks, 1 month, 2months, 3 months, 6 months, 1 year, 2 years, 5 years, or 10 years ormore previously. Aging-associated cognitive impairments includeimpairments in cognitive ability that are typically associated withaging, including, for example, cognitive impairment associated with thenatural aging process, e.g., mild cognitive impairment (M.C.I.); andcognitive impairment associated with an aging-associated disorder, thatis, a disorder that is seen with increasing frequency with increasingsenescence, e.g., a neurodegenerative condition such as Alzheimer'sdisease, Parkinson's disease, frontotemporal dementia, Huntington'sdisease, amyotrophic lateral sclerosis, multiple sclerosis, glaucoma,myotonic dystrophy, vascular dementia, and the like.

By “treatment” it is meant that at least an amelioration of one or moresymptoms associated with an aging-associated impairment afflicting theadult mammal is achieved, where amelioration is used in a broad sense torefer to at least a reduction in the magnitude of a parameter, e.g., asymptom associated with the impairment being treated. As such, treatmentalso includes situations where a pathological condition, or at leastsymptoms associated therewith, are completely inhibited, e.g., preventedfrom happening, or stopped, e.g., terminated, such that the adult mammalno longer suffers from the impairment, or at least the symptoms thatcharacterize the impairment. In some instances, “treatment”, “treating”and the like refer to obtaining a desired pharmacologic and/orphysiologic effect. The effect may be prophylactic in terms ofcompletely or partially preventing a disease or symptom thereof and/ormay be therapeutic in terms of a partial or complete cure for a diseaseand/or adverse effect attributable to the disease. “Treatment” may beany treatment of a disease in a mammal, and includes: (a) preventing thedisease from occurring in a subject which may be predisposed to thedisease but has not yet been diagnosed as having it; (b) inhibiting thedisease, i.e., arresting its development; or (c) relieving the disease,i.e., causing regression of the disease. Treatment may result in avariety of different physical manifestations, e.g., modulation in geneexpression, increased neurogenesis, rejuvenation of tissue or organs,etc. Treatment of ongoing disease, where the treatment stabilizes orreduces the undesirable clinical symptoms of the patient, occurs in someembodiments. Such treatment may be performed prior to complete loss offunction in the affected tissues. The subject therapy may beadministered during the symptomatic stage of the disease, and in somecases after the symptomatic stage of the disease.

In some instances where the aging-associated impairment isaging-associated cognitive decline, treatment by methods of the presentdisclosure slows, or reduces, the progression of aging-associatedcognitive decline. In other words, cognitive abilities in the individualdecline more slowly, if at all, following treatment by the disclosedmethods than prior to or in the absence of treatment by the disclosedmethods. In some instances, treatment by methods of the presentdisclosure stabilizes the cognitive abilities of an individual. Forexample, the progression of cognitive decline in an individual sufferingfrom aging-associated cognitive decline is halted following treatment bythe disclosed methods. As another example, cognitive decline in anindividual, e.g., an individual 40 years old or older, that is projectedto suffer from aging-associated cognitive decline, is preventedfollowing treatment by the disclosed methods. In other words, no(further) cognitive impairment is observed. In some instances, treatmentby methods of the present disclosure reduces, or reverses, cognitiveimpairment, e.g., as observed by improving cognitive abilities in anindividual suffering from aging-associated cognitive decline. In otherwords, the cognitive abilities of the individual suffering fromaging-associated cognitive decline following treatment by the disclosedmethods are better than they were prior to treatment by the disclosedmethods, i.e., they improve upon treatment. In some instances, treatmentby methods of the present disclosure abrogates cognitive impairment. Inother words, the cognitive abilities of the individual suffering fromaging-associated cognitive decline are restored, e.g., to their levelwhen the individual was about 40 years old or less, following treatmentby the disclosed methods, e.g., as evidenced by improved cognitiveabilities in an individual suffering from aging-associated cognitivedecline.

In some instances, treatment of an adult mammal in accordance with themethods results in a change in a central organ, e.g., a central nervoussystem organ, such as the brain, spinal cord, etc., where the change maymanifest in a number of different ways, e.g., as described in greaterdetail below, including but not limited to molecular, structural and/orfunctional, e.g., in the form of enhanced neurogenesis.

As summarized above, methods described herein are methods of treating anaging-associated impairment, e.g., as described above, in an adultmammal. By adult mammal is meant a mammal that has reached maturity,i.e., that is fully developed. As such, adult mammals are not juvenile.Mammalian species that may be treated with the present methods includecanines and felines; equines; bovines; ovines; etc., and primates,including humans. The subject methods, compositions, and reagents mayalso be applied to animal models, including small mammals, e.g., murine,lagomorpha, etc., for example, in experimental investigations. Thediscussion below will focus on the application of the subject methods,compositions, reagents, devices and kits to humans, but it will beunderstood by the ordinarily skilled artisan that such descriptions canbe readily modified to other mammals of interest based on the knowledgein the art.

The age of the adult mammal may vary, depending on the type of mammalthat is being treated. Where the adult mammal is a human, the age of thehuman is generally 18 years or older. In some instances, the adultmammal is an individual suffering from or at risk of suffering from anaging-associated impairment, such as an aging-associated cognitiveimpairment, where the adult mammal may be one that has been determined,e.g., in the form of receiving a diagnosis, to be suffering from or atrisk of suffering from an aging-associated impairment, such as anaging-associated cognitive impairment. The phrase “an individualsuffering from or at risk of suffering from an aging-associatedcognitive impairment” refers to an individual that is about 50 years oldor older, e.g., 60 years old or older, 70 years old or older, 80 yearsold or older, and sometimes no older than 100 years old, such as 90years old, i.e., between the ages of about 50 and 100, e.g., 50, 55, 60,65, 70, 75, 80, 85 or about 90 years old. The individual may suffer froman aging associated condition, e.g., cognitive impairment, associatedwith the natural aging process, e.g., M.C.I. Alternatively, theindividual may be 50 years old or older, e.g., 60 years old or older, 70years old or older, 80 years old or older, 90 years old or older, andsometimes no older than 100 years old, i.e., between the ages of about50 and 100, e.g., 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or about 100years old, and has not yet begun to show symptoms of an aging associatedcondition, e.g., cognitive impairment. In yet other embodiments, theindividual may be of any age where the individual is suffering from acognitive impairment due to an aging-associated disease, e.g.,Alzheimer's disease, Parkinson's disease, frontotemporal dementia,Huntington's disease, amyotrophic lateral sclerosis, multiple sclerosis,glaucoma, myotonic dystrophy, dementia, and the like. In some instances,the individual is an individual of any age that has been diagnosed withan aging-associated disease that is typically accompanied by cognitiveimpairment, e.g., Alzheimer's disease, Parkinson's disease,frontotemporal dementia, progressive supranuclear palsy, Huntington'sdisease, amyotrophic lateral sclerosis, spinal muscular atrophy,multiple sclerosis, multi-system atrophy, glaucoma, ataxias, myotonicdystrophy, dementia, and the like, where the individual has not yetbegun to show symptoms of cognitive impairment.

As summarized above, aspects of the methods include reducing theβ2-microglobulin (B2M) level in the mammal in a manner sufficient totreat the aging impairment in the mammal, e.g., as described above. Byreducing the B2M level is meant lowering the amount of B2M in themammal, such as the amount of extracellular B2M in the mammal. While themagnitude of the reduction may vary, in some instances the magnitude is2-fold or greater, such as 5-fold or greater, including 10-fold orgreater, e.g., 15-fold or greater, 20-fold or greater, 25-fold orgreater (as compared to a suitable control), where in some instances themagnitude is such that the amount of detectable free B2M in thecirculatory system of the individual is 50% or less, such as 25% orless, including 10% or less, e.g., 1% or less, relative to the amountthat was detectable prior to intervention according to the invention,and in some instances the amount is undetectable following intervention.

The B2M level may be reduced using any convenient protocol. In someinstances, the B2M level is reduced by removing systemic B2M from theadult mammal, e.g., by removing B2M from the circulatory system of theadult mammal. In such instances, any convenient protocol for removingcirculatory B2M may be employed. For example, blood may be obtained fromthe adult mammal and extra-corporeally processed to remove B2M from theblood to produce B2M depleted blood, which resultant B2M depleted bloodmay then be returned to the adult mammal. Such protocols may employ avariety of different techniques in order to remove B2M from the obtainedblood. For example, the obtained blood may be contacted with a filteringcomponent, e.g., a membrane, etc., which allows passage of B2M butinhibits passage of other blood components, e.g., cells, etc. In someinstances, the obtained blood may be contacted with a B2M absorptivecomponent, e.g., porous bead or particulate composition, which absorbsB2M from the blood. In yet other instances, the obtained blood may becontacted with a B2M binding member stably associated with a solidsupport, such that B2M binds to the binding member and is therebyimmobilized on the solid support, thereby providing for separation ofB2M from other blood constituents. The protocol employed may or may notbe configured to selectively remove B2M from the obtained blood, asdesired. A number of different technologies are known for removing B2Mfrom blood, and may be employed in embodiments of the invention, wheresuch technologies include those described in U.S. Pat. Nos. 4,872,983;5,240,614; 6,416,487; 6,419,830; 6,423,024; 6,855,121; 7,066,900;8,211,310; 8,349,550; as well as published United States PatentApplication Publication No. 20020143283 and published PCT ApplicationPublication Nos.: WO/1999/006098 and WO/2003/020403; the disclosures ofwhich applications are herein incorporated by reference.

In some embodiments, the B2M level is reduced by administering to themammal an effective amount of a B2M level reducing agent. As such, inpracticing methods according to these embodiments of the invention, aneffective amount of the active agent, e.g., B2M modulatory agent, isprovided to the adult mammal.

Depending on the particular embodiments being practiced, a variety ofdifferent types of active agents may be employed. In some instances, theagent modulates expression of the RNA and/or protein from the gene, suchthat it changes the expression of the RNA or protein from the targetgene in some manner. In these instances, the agent may change expressionof the RNA or protein in a number of different ways. In certainembodiments, the agent is one that reduces, including inhibits,expression of a B2M protein. Inhibition of B2M protein expression may beaccomplished using any convenient means, including use of an agent thatinhibits B2M protein expression, such as, but not limited to: RNAiagents, antisense agents, agents that interfere with a transcriptionfactor binding to a promoter sequence of the B2M gene, or inactivationof the B2M gene, e.g., through recombinant techniques, etc.

For example, the transcription level of a B2M protein can be regulatedby gene silencing using RNAi agents, e.g., double-strand RNA (see e.g.,Sharp, Genes and Development (1999) 13: 139-141). RNAi, such asdouble-stranded RNA interference (dsRNAi) or small interfering RNA(siRNA), has been extensively documented in the nematode C. elegans(Fire, et al, Nature (1998) 391:806-811) and routinely used to “knockdown” genes in various systems. RNAi agents may be dsRNA or atranscriptional template of the interfering ribonucleic acid which canbe used to produce dsRNA in a cell. In these embodiments, thetranscriptional template may be a DNA that encodes the interferingribonucleic acid. Methods and procedures associated with RNAi are alsodescribed in published PCT Application Publication Nos. WO 03/010180 andWO 01/68836, the disclosures of which applications are incorporatedherein by reference. dsRNA can be prepared according to any of a numberof methods that are known in the art, including in vitro and in vivomethods, as well as by synthetic chemistry approaches. Examples of suchmethods include, but are not limited to, the methods described by Sadheret al., Biochem. Int. (1987) 14:1015; Bhattacharyya, Nature (1990)343:484; and U.S. Pat. No. 5,795,715, the disclosures of which areincorporated herein by reference. Single-stranded RNA can also beproduced using a combination of enzymatic and organic synthesis or bytotal organic synthesis. The use of synthetic chemical methods enableone to introduce desired modified nucleotides or nucleotide analogs intothe dsRNA. dsRNA can also be prepared in vivo according to a number ofestablished methods (see, e.g., Sambrook, et al. (1989) MolecularCloning: A Laboratory Manual, 2nd ed.; Transcription and Translation (B.D. Hames, and S. J. Higgins, Eds., 1984); DNA Cloning, volumes I and II(D. N. Glover, Ed., 1985); and Oligonucleotide Synthesis (M. J. Gait,Ed., 1984, each of which is incorporated herein by reference). A numberof options can be utilized to deliver the dsRNA into a cell orpopulation of cells such as in a cell culture, tissue, organ or embryo.For instance, RNA can be directly introduced intracellularly. Variousphysical methods are generally utilized in such instances, such asadministration by microinjection (see, e.g., Zernicka-Goetz, et al.Development (1997)124:1133-1137; and Wianny, et al., Chromosoma (1998)107: 430-439). Other options for cellular delivery includepermeabilizing the cell membrane and electroporation in the presence ofthe dsRNA, liposome-mediated transfection, or transfection usingchemicals such as calcium phosphate. A number of established genetherapy techniques can also be utilized to introduce the dsRNA into acell. By introducing a viral construct within a viral particle, forinstance, one can achieve efficient introduction of an expressionconstruct into the cell and transcription of the RNA encoded by theconstruct. Specific examples of RNAi agents that may be employed toreduce B2M expression include, but are not limited to: dsRNA and shortinterfering RNA (siRNA) corresponding to B2M with the following senseand antisense sequences (sense) 5′-GAUUCAGGUUUACUCACGUdTdT-3′ (SEQ IDNO:01) and (antisense) 5′-ACGUGAGUAAACCUGAAUCdTdT-3′ (SEQ ID NO:02)(asdescribed in Matin, et al., “Specific knockdown of Oct4 andbeta2-microglobulin expression by RNA interference in human embryonicstem cells and embryonic carcinoma cells,” Stem Cells (2004) 22: 659-68)and WO/2004/085654; shRNA (GCCACTCCCACCCTTTCTCAT)(SEQ ID NO:03) (asdisclosed in Goyos, et al., “Involvement of nonclassical MHC class Ibmolecules in heat shock protein-mediated anti-tumor responses,” (2007)37: 1494-501); as well as the RNAi agents disclosed in Figueiredo, etal., “Generation of HLA-deficient platelets from hematopoieticprogenitor cells,” Transfusion (2010) 50: 1690-701. Bhatt, et al.,“Knockdown of beta2-microglobulin perturbs the subcellular distributionof HFE and hepcidin,” Biochemical and Biophysical ResearchCommunications (2009) 378: 727-31, Elders, et al., “Targeted knockdownof canine KIT (stem cell factor receptor) using RNA interference,”Veterinary Immunology and Immunopathology (2011) 141:151-6, Heikkila, etal., “Internalization of coxsackievirus A9 is mediated by beta2-microglobulin, dynamin, and Arf6 but not by caveolin-1 or clathrin,”(2010) 84: 3666-81, Figueiredo, et al., “Class-, gene-, andgroup-specific HLA silencing by lentiviral shRNA delivery (2006) 84:425-37, WO/2004/020586, US20040127445 and US20130096370.

In some instances, antisense molecules can be used to down-regulateexpression of a B2M gene in the cell. The anti-sense reagent may beantisense oligodeoxynucleotides (ODN), particularly synthetic ODN havingchemical modifications from native nucleic acids, or nucleic acidconstructs that express such anti-sense molecules as RNA. The antisensesequence is complementary to the mRNA of the targeted protein, andinhibits expression of the targeted protein. Antisense molecules inhibitgene expression through various mechanisms, e.g., by reducing the amountof mRNA available for translation, through activation of RNAse H, orsteric hindrance. One or a combination of antisense molecules may beadministered, where a combination may include multiple differentsequences.

Antisense molecules may be produced by expression of all or a part ofthe target gene sequence in an appropriate vector, where thetranscriptional initiation is oriented such that an antisense strand isproduced as an RNA molecule. Alternatively, the antisense molecule is asynthetic oligonucleotide. Antisense oligonucleotides will generally beat least about 7, usually at least about 12, more usually at least about20 nucleotides in length, and not more than about 500, usually not morethan about 50, more usually not more than about 35 nucleotides inlength, where the length is governed by efficiency of inhibition,specificity, including absence of cross-reactivity, and the like. Shortoligonucleotides, of from 7 to 8 bases in length, can be strong andselective inhibitors of gene expression (see Wagner et al., NatureBiotechnol. (1996)14:840-844).

A specific region or regions of the endogenous sense strand mRNAsequence are chosen to be complemented by the antisense sequence.Selection of a specific sequence for the oligonucleotide may use anempirical method, where several candidate sequences are assayed forinhibition of expression of the target gene in an in vitro or animalmodel. A combination of sequences may also be used, where severalregions of the mRNA sequence are selected for antisense complementation.

Antisense oligonucleotides may be chemically synthesized by methodsknown in the art (see Wagner et al. (1993), supra.) Oligonucleotides maybe chemically modified from the native phosphodiester structure, inorder to increase their intracellular stability and binding affinity. Anumber of such modifications have been described in the literature,which alter the chemistry of the backbone, sugars or heterocyclic bases.Among useful changes in the backbone chemistry are phosphorothioates;phosphorodithioates, where both of the non-bridging oxygens aresubstituted with sulfur; phosphoroamidites; alkyl phosphotriesters andboranophosphates. Achiral phosphate derivatives include3′-O-5′-S-phosphorothioate, 3′-S-5′-O-phosphorothioate,3′-CH.sub.2-5′-O-phosphonate and 3′-NH-5′-O-phosphoroamidate. Peptidenucleic acids replace the entire ribose phosphodiester backbone with apeptide linkage. Sugar modifications are also used to enhance stabilityand affinity. The α-anomer of deoxyribose may be used, where the base isinverted with respect to the natural β-anomer. The 2′-OH of the ribosesugar may be altered to form 2′-O-methyl or 2′-O-allyl sugars, whichprovides resistance to degradation without comprising affinity.Modification of the heterocyclic bases must maintain proper basepairing. Some useful substitutions include deoxyuridine fordeoxythymidine; 5-methyl-2′-deoxycytidine and 5-bromo-2′-deoxycytidinefor deoxycytidine. 5-propynyl-2′-deoxyuridine and5-propynyl-2′-deoxycytidine have been shown to increase affinity andbiological activity when substituted for deoxythymidine anddeoxycytidine, respectively. Specific examples of antisense agents thatmay be employed to reduce B2M expression include, but are not limitedto:

Code Oligonucleotide MB-00027βA*βG*dT*dT*dG*dC*dC*dA*dG*dC*dC*dC*dT*βZ*βZ MB-00540Eru*SS*βA*βG*dT*dT*dG*dC*dC*dA*dG*dC*dC*dC*dT*βZ*βZ MB-00541Myr*SS*βA*βG*dT*dT*dG*dC*dC*dA*dG*dC*dC*dC*dT*βZ*βZ MB-00542Dier*SS*βA*βG*dT*dT*dG*dC*dC*dA*dG*dC*dC*dC*dT*βZ*βZ MB-00543Ermy*SS*βA*βG*dT*dT*dG*dC*dC*dA*dG*dC*dC*dC*dT*βZ*βZ

(SEQ ID NOS: 04 to 08)

as described in WO/2004/004575; as well as those antisense agentsdescribed in: Lichtenstein, et al., “Effects of beta-2 microglobulinanti-sense oligonucleotides on sensitivity of HER2/neuoncogene-expressing and nonexpressing target cells tolymphocyte-mediated lysis,” Cell Immunology (1992) 141: 219-32,Ogretmen, et al., “Molecular mechanisms of loss of beta 2-microglobulinexpression in drug-resistant breast cancer sublines and its involvementin drug resistance,” Biochemistry (1998) 37: 11679-91, WO/2004/020586;WO/2006/130949; U.S. Pat. Nos. 7,553,484; and 8,715,654.

As an alternative to anti-sense inhibitors, catalytic nucleic acidcompounds, e.g. ribozymes, anti-sense conjugates, etc. may be used toinhibit gene expression. Ribozymes may be synthesized in vitro andadministered to the patient, or may be encoded on an expression vector,from which the ribozyme is synthesized in the targeted cell (forexample, see International patent application WO 9523225, and Beigelmanet al. Nucl. Acids Res. (1995) 23:4434-42). Examples of oligonucleotideswith catalytic activity are described in WO 9506764. Conjugates ofanti-sense ODN with a metal complex, e.g. terpyridylCu(II), capable ofmediating mRNA hydrolysis are described in Bashkin at al. Appl. Biochem.Biotechnol. (1995) 54:43-56.

In another embodiment, the B2M gene is inactivated so that it no longerexpresses a functional protein. By inactivated is meant that the gene,e.g., coding sequence and/or regulatory elements thereof, is geneticallymodified so that it no longer expresses a functional B2M protein, e.g.,at least with respect to B2M aging impairment activity. The alterationor mutation may take a number of different forms, e.g., through deletionof one or more nucleotide residues, through exchange of one or morenucleotide residues, and the like. One means of making such alterationsin the coding sequence is by homologous recombination. Methods forgenerating targeted gene modifications through homologous recombinationare known in the art, including those described in: U.S. Pat. Nos.6,074,853; 5,998,209; 5,998,144; 5,948,653; 5,925,544; 5,830,698;5,780,296; 5,776,744; 5,721,367; 5,614.396; 5,612.205; the disclosuresof which are herein incorporated by reference.

Also of interest in certain embodiments are dominant negative mutants ofB2M proteins, where expression of such mutants in the cell result in amodulation, e.g., decrease, in B2M mediated aging impairment. Dominantnegative mutants of B2M are mutant proteins that exhibit dominantnegative B2M activity. As used herein, the term “dominant-negative B2Mactivity” or “dominant negative activity” refers to the inhibition,negation, or diminution of certain particular activities of B2M, andspecifically to B2M mediated aging impairment. Dominant negativemutations are readily generated for corresponding proteins. These mayact by several different mechanisms, including mutations in asubstrate-binding domain; mutations in a catalytic domain; mutations ina protein binding domain (e.g., multimer forming, effector, oractivating protein binding domains); mutations in cellular localizationdomain, etc. A mutant polypeptide may interact with wild-typepolypeptides (made from the other allele) and form a non-functionalmultimer. In certain embodiments, the mutant polypeptide will beoverproduced. Point mutations are made that have such an effect. Inaddition, fusion of different polypeptides of various lengths to theterminus of a protein, or deletion of specific domains can yielddominant negative mutants. General strategies are available for makingdominant negative mutants (see for example, Herskowitz. Nature (1987)329:219, and the references cited above). Such techniques are used tocreate loss of function mutations, which are useful for determiningprotein function. Methods that are well known to those skilled in theart can be used to construct expression vectors containing codingsequences and appropriate transcriptional and translational controlsignals for increased expression of an exogenous gene introduced into acell. These methods include, for example, in vitro recombinant DNAtechniques, synthetic techniques, and in vivo genetic recombination.Alternatively, RNA capable of encoding gene product sequences may bechemically synthesized using, for example, synthesizers. See, forexample, the techniques described in “Oligonucleotide Synthesis”, 1984,Gait, M. J. ed., IRL Press, Oxford.

In yet other embodiments, the agent is an agent that modulates, e.g.,inhibits, B2M activity by binding to B2M and/or inhibiting binding ofB2M to a second protein, e.g., a protein member of MHC1. For example,small molecules that bind to the B2M and inhibit its activity are ofinterest. Naturally occurring or synthetic small molecule compounds ofinterest include numerous chemical classes, such as organic molecules,e.g., small organic compounds having a molecular weight of more than 50and less than about 2,500 daltons. Candidate agents comprise functionalgroups for structural interaction with proteins, particularly hydrogenbonding, and typically include at least an amine, carbonyl, hydroxyl orcarboxyl group, preferably at least two of the functional chemicalgroups. The candidate agents may include cyclical carbon or heterocyclicstructures and/or aromatic or polyaromatic structures substituted withone or more of the above functional groups. Candidate agents are alsofound among biomolecules including peptides, saccharides, fatty acids,steroids, purines, pyrimidines, derivatives, structural analogs orcombinations thereof. Such molecules may be identified, among otherways, by employing the screening protocols described below. Specificexamples of small molecule agents agents that may be employed to reduceB2M expression include, but are not limited to: Riamycin SV:(7S,9E,11S,12R,13S,14R,15R,16R,17S,18S,19E,21Z)-2,15,17,27,29-pentahydroxy-11-methoxy-3,7,12,14,16,18,22-heptamethyl-26-{(E)-[(4-methylpiperazin-1-yl)imino]methyl}-6,23-dioxo-8,30-dioxa-24-azatetracyclo[23.3.1.14,7.05,28]triaconta-1(28),2,4,9,19,21,25(29),26-octaen-13-ylacetate (as disclosed in Woods, et al., “Ligand binding to distinctstates diverts aggregation of an amyloid-forming protein” NatureChemical Biology (2011) 7: 730-9); meclocycline, doxycycline,4-epi-oxytetracylcine, rolitetracycline, anhydrochlortetracycline,methacycline and oxytetracycline (as described in Giorgetti, et al.,“Effect of tetracyclines on the dynamics of formation anddestructuration of beta2-microglobulin amyloid fibrils,” The Journal ofBiological Chemistry (2011) 286: 2121-31); peptides D-TLKIVW, D-TWKLVL,D-YVIIER and D-DYYFEF (as described in U.S. Pat. No. 8,754,034); as wellas the agents described in: Morozov, et al., “Survey of small moleculeand ion binding to beta 2-microglobulin-possible relation to BEN,”(1991) 34: S85-8, Regazzoni, et al., “Screening of fibrillogenesisinhibitors of B2-microglobulin: integrated strategies by massspectrometry capillary electrophoresis and in silico simulations,”Analytica Chimica Acta (2011) 685: 153-61, Quaglia, et al., “Search ofligands for the amyloidogenic protein beta2-microglobulin by capillaryelectrophoresis and other techniques,” Electrophoresis (2005) 26:4055-63, Ozawa, et al., “Inhibition of beta2-microglobulin amyloidfibril formation by alpha2-macroglobulin,” The Journal of BiologicalChemistry (2011) 286: 9668-9676, Pullara and Emanuele, “Early stages ofbeta2-microglobulin aggregation and the inhibiting action ofalphaB-crystallin,” (2008) 73: 1037-46, Wanchu, et al., “Suppression ofbeta 2 microglobulin by pentoxiphylline therapy in asymptomatic HIVinfected individuals,” (2001) 113: 75-7. Brancolini, et al., “Can smallhydrophobic gold nanoparticles inhibit B2-microglobulin fibrillation?,”Nanoscale (2014) 6: 7903-11, US20040127445 and US20130331327.

In certain embodiments, the administered active agent is a B2M specificbinding member. In general, useful B2M specific binding members exhibitan affinity (Kd) for a target B2M, such as human B2M, that is sufficientto provide for the desired reduction in aging associated impairment B2Mactivity. As used herein, the term “affinity” refers to the equilibriumconstant for the reversible binding of two agents; “affinity” can beexpressed as a dissociation constant (Kd). Affinity can be at least1-fold greater, at least 2-fold greater, at least 3-fold greater, atleast 4-fold greater, at least 5-fold greater, at least 6-fold greater,at least 7-fold greater, at least 8-fold greater, at least 9-foldgreater, at least 10-fold greater, at least 20-fold greater, at least30-fold greater, at least 40-fold greater, at least 50-fold greater, atleast 60-fold greater, at least 70-fold greater, at least 80-foldgreater, at least 90-fold greater, at least 100-fold greater, or atleast 1000-fold greater, or more, than the affinity of an antibody forunrelated amino acid sequences. Affinity of a specific binding member toa target protein can be, for example, from about 100 nanomolar (nM) toabout 0.1 nM, from about 100 nM to about 1 picomolar (pM), or from about100 nM to about 1 femtomolar (fM) or more. The term “binding” refers toa direct association between two molecules, due to, for example,covalent, electrostatic, hydrophobic, and ionic and/or hydrogen-bondinteractions, including interactions such as salt bridges and waterbridges. In some embodiments, the antibodies bind human B2M withnanomolar affinity or picomolar affinity. In some embodiments, theantibodies bind human B2M with a Kd of less than about 100 nM, 50 nM, 20nM, 20 nM, or 1 nM.

Examples of B2M specific binding members include B2M antibodies andbinding fragments thereof. Non-limiting examples of such antibodiesinclude antibodies directed against any epitope of B2M. Also encompassedare bispecific antibodies, i.e., antibodies in which each of the twobinding domains recognizes a different binding epitope. The amino acidsequence of human B2M is disclosed in Cunningham, et al., “The completeamino acid sequence of beta-2-microglobulin,” Biochemistry (1973) 12:4811-4821.

Antibody specific binding members that may be employed include fullantibodies or immunoglobulins of any isotype, as well as fragments ofantibodies which retain specific binding to antigen, including, but notlimited to, Fab, Fv, scFv, and Fd fragments, chimeric antibodies,humanized antibodies, single-chain antibodies, and fusion proteinscomprising an antigen-binding portion of an antibody and a non-antibodyprotein. The antibodies may be detectably labeled, e.g., with aradioisotope, an enzyme which generates a detectable product, afluorescent protein, and the like. The antibodies may be furtherconjugated to other moieties, such as members of specific binding pairs,e.g., biotin (member of biotin-avidin specific binding pair), and thelike. Also encompassed by the term are Fab′, Fv, F(ab′)2, and or otherantibody fragments that retain specific binding to antigen, andmonoclonal antibodies. An antibody may be monovalent or bivalent.

“Antibody fragments” comprise a portion of an intact antibody, forexample, the antigen binding or variable region of the intact antibody.Examples of antibody fragments include Fab, Fab′, F(ab′)2, and Fvfragments; diabodies; linear antibodies (Zapata et al., Protein Eng.8(10): 1057-1062 (1995)); single-chain antibody molecules; andmultispecific antibodies formed from antibody fragments. Papaindigestion of antibodies produces two identical antigen-bindingfragments, called “Fab” fragments, each with a single antigen-bindingsite, and a residual “Fc” fragment, a designation reflecting the abilityto crystallize readily. Pepsin treatment yields an F(ab′)2 fragment thathas two antigen combining sites and is still capable of cross-linkingantigen.

“Fv” is the minimum antibody fragment which contains a completeantigen-recognition and -binding site. This region consists of a dimerof one heavy- and one light-chain variable domain in tight, non-covalentassociation. It is in this configuration that the three CDRS of eachvariable domain interact to define an antigen-binding site on thesurface of the VH-VL dimer. Collectively, the six CDRs conferantigen-binding specificity to the antibody. However, even a singlevariable domain (or half of an Fv comprising only three CDRs specificfor an antigen) has the ability to recognize and bind antigen, althoughat a lower affinity than the entire binding site.

The “Fab” fragment also contains the constant domain of the light chainand the first constant domain (CH1) of the heavy chain. Fab fragmentsdiffer from Fab′ fragments by the addition of a few residues at thecarboxyl terminus of the heavy chain CH1 domain including one or morecysteines from the antibody hinge region. Fab′-SH is the designationherein for Fab′ in which the cysteine residue(s) of the constant domainsbear a free thiol group. F(ab′)2 antibody fragments originally wereproduced as pairs of Fab′ fragments which have hinge cysteines betweenthem. Other chemical couplings of antibody fragments are also known.

The “light chains” of antibodies (immunoglobulins) from any vertebratespecies can be assigned to one of two clearly distinct types, calledkappa and lambda, based on the amino acid sequences of their constantdomains. Depending on the amino acid sequence of the constant domain oftheir heavy chains, immunoglobulins can be assigned to differentclasses. There are five major classes of immunoglobulins: IgA, IgD, IgE,IgG, and IgM, and several of these may be further divided intosubclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2.

“Single-chain Fv” or “sFv” antibody fragments comprise the VH and VLdomains of antibody, wherein these domains are present in a singlepolypeptide chain. In some embodiments, the Fv polypeptide furthercomprises a polypeptide linker between the VH and VL domains, whichenables the sFv to form the desired structure for antigen binding. For areview of sFv, see Pluckthun in The Pharmacology of MonoclonalAntibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, NewYork, pp. 269-315 (1994).

Antibodies that may be used in connection with the present disclosurethus can encompass monoclonal antibodies, polyclonal antibodies,bispecific antibodies, Fab antibody fragments, F(ab)2 antibodyfragments, Fv antibody fragments (e.g., VH or VL), single chain Fvantibody fragments and dsFv antibody fragments. Furthermore, theantibody molecules may be fully human antibodies, humanized antibodies,or chimeric antibodies. In some embodiments, the antibody molecules aremonoclonal, fully human antibodies.

The antibodies that may be used in connection with the presentdisclosure can include any antibody variable region, mature orunprocessed, linked to any immunoglobulin constant region. If a lightchain variable region is linked to a constant region, it can be a kappachain constant region. If a heavy chain variable region is linked to aconstant region, it can be a human gamma 1, gamma 2, gamma 3 or gamma 4constant region, more preferably, gamma 1, gamma 2 or gamma 4 and evenmore preferably gamma 1 or gamma 4.

In some embodiments, fully human monoclonal antibodies directed againstB2M are generated using transgenic mice carrying parts of the humanimmune system rather than the mouse system.

Minor variations in the amino acid sequences of antibodies orimmunoglobulin molecules are encompassed by the present invention,providing that the variations in the amino acid sequence maintain atleast 75%, e.g., at least 80%, 90%, 95%, or 99% of the sequence. Inparticular, conservative amino acid replacements are contemplated.Conservative replacements are those that take place within a family ofamino acids that are related in their side chains. Whether an amino acidchange results in a functional peptide can readily be determined byassaying the specific activity of the polypeptide derivative. Fragments(or analogs) of antibodies or immunoglobulin molecules, can be readilyprepared by those of ordinary skill in the art. Preferred amino- andcarboxy-termini of fragments or analogs occur near boundaries offunctional domains. Structural and functional domains can be identifiedby comparison of the nucleotide and/or amino acid sequence data topublic or proprietary sequence databases. Preferably, computerizedcomparison methods are used to identify sequence motifs or predictedprotein conformation domains that occur in other proteins of knownstructure and/or function. Methods to identify protein sequences thatfold into a known three-dimensional structure are known. Sequence motifsand structural conformations may be used to define structural andfunctional domains in accordance with the invention.

Specific examples of antibody agents that may be employed to reduce B2Mexpression include, but are not limited to: Anti-B2m B1-1G6(immunoglobulin G2a [IgG2]), B2-62-2 (IgG2a), and C21-48A (IgG2b) fromImmunotech S.A. (Marseille, France); Anti-B2m MAb HC11-151-1 (IgG1) (asdisclosed in Corbeau, et al., “An early postinfection signal mediated bymonocional anti-beta 2 microglobulin antibody is responsible for delayedproduction of human immunodeficiency virus type 1 in peripheral bloodmononuclear cells,” Journal of Virology (1990) 64: 1459-64); clone B2,mouse IgG1 (Sero-tec Ltd., Oxford, UK); mouse mAbs against human B2M asdisclosed in Yang, et al., “Targeting beta(2)-microglobulin forinduction of tumor apoptosis in human hematological malignancies,”(2006) 10: 295-307; 1B749 (IgG2a) and HB28 (IgG2b) (as disclosed inPokrass, et al., “Activation of complement by monoclonal antibodies thattarget cell-associated B2-microglobulin: implications for cancerimmunotherapy,” (2013) 56: 549-60); anti-B2-microglobulin (BBM.1antibody) (as disclosed in Brodsky, et al., “Characterization of amonoclonal anti-beta 2-microglobulin antibody and its use in the geneticand biochemical analysis of major histocompatibility antigens,” EuropeanJournal of Immunology (1979) 9: 536-45; BBM-1 (as disclosed inKorkolopoulou, “Loss of antigen-presenting molecules (MHC class I andTAP-1) in lung cancer,” British Journal of Cancer (1996) 73: 148-53;B1.1G6, C23.24.2, B2.62.2, and C21.48A1 antibodies (as disclosed inLiabeuf, et al., “An antigenic determinant of human beta 2-microglobulinmasked by the association with HLA heavy chains at the cell surface:analysis using monoclonal antibodies,” Journal of Immunology (1981) 127:1542-8); as well as those antibody agents described in: Zhang, et al.,“Anti-B2M monoclonal antibodies kill myeloma cells via cell- andcomplement-mediated cytotoxicity,” International Journal of Cancer(2014) 135: 1132-41, Yang, at al., “Anti beta2-microglobulin monoclonalantibodies induce apoptosis in myeloma cells by recruiting MHC class Ito and excluding growth and survival cytokine receptors from lipidrafts,” Blood (2007) 110: 3028-35, Josson, et al., “Inhibition ofB2-microglobulin/hemochromatosis enhances radiation sensitivity byinduction of iron overload in prostate cancer cells,” (2013) 8: e68366,Par and Falus, “Serum beta 2-microglobulin (beta 2m) and anti-beta 2mantibody in chronic hepatitis,” Acta Medica Hungarica (1986) 43:343-9,Huang, et al., “Androgen receptor survival signaling is blocked byanti-beta2-microglobulin monoclonal antibody via a MAPK/lipogenicpathway in human prostate cancer cells,” The Journal of BiologicalChemistry (2010) 285: 7947-56, Tam and Messner, “Differential inhibitionof mitogenic responsiveness by monoclonal antibodies to beta2-microglobulin,” (1991) 133: 219-33, Domanska, et al., “Atomicstructure of a nanobody-trapped domain-swapped dimer of an amyloidogenicbeta2-microglobulin variant,” Proc Natl Acad Sci USA. (2011)108(4):1314-9, Falus, et al., “Prevalence of anti-beta-2 microglobulinautoantibodies in sera of rheumatoid arthritis patients withextra-articular manifestations,” Annals of the Rheumatic Diseases,(1981) 40: 409-413, Shabunina, et al., “Immunosorbent for Removal ofB2-microglobulin from Human Blood Plasma,” Bulletin of ExperimentalBiology and Medicine (2001) 132: 984-986), WO/2010/017443, U.S. Pat. No.7,341,721, WO/1996/002278, WO/2003/079023, and WO/1990/013657.

In those embodiments where an active agent is administered to the adultmammal, the active agent(s) may be administered to the adult mammalusing any convenient administration protocol capable of resulting in thedesired activity. Thus, the agent can be incorporated into a variety offormulations, e.g., pharmaceutically acceptable vehicles, fortherapeutic administration. More particularly, the agents of the presentinvention can be formulated into pharmaceutical compositions bycombination with appropriate, pharmaceutically acceptable carriers ordiluents, and may be formulated into preparations in solid, semi-solid,liquid or gaseous forms, such as tablets, capsules, powders, granules,ointments (e.g., skin creams), solutions, suppositories, injections,inhalants and aerosols. As such, administration of the agents can beachieved in various ways, including oral, buccal, rectal, parenteral,intraperitoneal, intradermal, transdermal, intracheal, etc.,administration.

In pharmaceutical dosage forms, the agents may be administered in theform of their pharmaceutically acceptable salts, or they may also beused alone or in appropriate association, as well as in combination,with other pharmaceutically active compounds. The following methods andexcipients are merely exemplary and are in no way limiting.

For oral preparations, the agents can be used alone or in combinationwith appropriate additives to make tablets, powders, granules orcapsules, for example, with conventional additives, such as lactose,mannitol, corn starch or potato starch; with binders, such ascrystalline cellulose, cellulose derivatives, acacia, corn starch orgelatins; with disintegrators, such as corn starch, potato starch orsodium carboxymethylcellulose; with lubricants, such as talc ormagnesium stearate; and if desired, with diluents, buffering agents,moistening agents, preservatives and flavoring agents.

The agents can be formulated into preparations for injection bydissolving, suspending or emulsifying them in an aqueous or nonaqueoussolvent, such as vegetable or other similar oils, synthetic aliphaticacid glycerides, esters of higher aliphatic acids or propylene glycol;and if desired, with conventional additives such as solubilizers,isotonic agents, suspending agents, emulsifying agents, stabilizers andpreservatives.

The agents can be utilized in aerosol formulation to be administered viainhalation. The compounds of the present invention can be formulatedinto pressurized acceptable propellants such as dichlorodifluoromethane,propane, nitrogen and the like.

Furthermore, the agents can be made into suppositories by mixing with avariety of bases such as emulsifying bases or water-soluble bases. Thecompounds of the present invention can be administered rectally via asuppository. The suppository can include vehicles such as cocoa butter,carbowaxes and polyethylene glycols, which melt at body temperature, yetare solidified at room temperature.

Unit dosage forms for oral or rectal administration such as syrups,elixirs, and suspensions may be provided wherein each dosage unit, forexample, teaspoonful, tablespoonful, tablet or suppository, contains apredetermined amount of the composition containing one or moreinhibitors. Similarly, unit dosage forms for injection or intravenousadministration may comprise the inhibitor(s) in a composition as asolution in sterile water, normal saline or another pharmaceuticallyacceptable carrier.

The term “unit dosage form,” as used herein, refers to physicallydiscrete units suitable as unitary dosages for human and animalsubjects, each unit containing a predetermined quantity of compounds ofthe present invention calculated in an amount sufficient to produce thedesired effect in association with a pharmaceutically acceptablediluent, carrier or vehicle. The specifications for the novel unitdosage forms of the present invention depend on the particular compoundemployed and the effect to be achieved, and the pharmacodynamicsassociated with each compound in the host.

The pharmaceutically acceptable excipients, such as vehicles, adjuvants,carriers or diluents, are readily available to the public. Moreover,pharmaceutically acceptable auxiliary substances, such as pH adjustingand buffering agents, tonicity adjusting agents, stabilizers, wettingagents and the like, are readily available to the public.

Where the agent is a polypeptide, polynucleotide, analog or mimeticthereof, it may be introduced into tissues or host cells by any numberof routes, including viral infection, microinjection, or fusion ofvesicles. Jet injection may also be used for intramuscularadministration, as described by Furth et al., Anal Biochem. (1992)205:365-368. The DNA may be coated onto gold microparticles, anddelivered intradermally by a particle bombardment device, or “gene gun”as described in the literature (see, for example, Tang et al., Nature(1992) 356:152-154), where gold microprojectiles are coated with theDNA, then bombarded into skin cells. For nucleic acid therapeuticagents, a number of different delivery vehicles find use, includingviral and non-viral vector systems, as are known in the art.

Those of skill in the art will readily appreciate that dose levels canvary as a function of the specific compound, the nature of the deliveryvehicle, and the like. Preferred dosages for a given compound arereadily determinable by those of skill in the art by a variety of means.

In those embodiments where an effective amount of an active agent isadministered to the adult mammal, the amount or dosage is effective whenadministered for a suitable period of time, such as one week or longer,including two weeks or longer, such as 3 weeks or longer, 4 weeks orlonger, 8 weeks or longer, etc., so as to evidence a reduction in theimpairment, e.g., cognition decline and/or cognitive improvement in theadult mammal. For example, an effective dose is the dose that, whenadministered for a suitable period of time, such as at least about oneweek, and maybe about two weeks, or more, up to a period of about 3weeks, 4 weeks, 8 weeks, or longer, will slow e.g., by about 20% ormore, e.g., by 30% or more, by 40% or more, or by 50% or more, in someinstances by 60% or more, by 70% or more, by 80% or more, or by 90% ormore, e.g., will halt, cognitive decline in a patient suffering fromnatural aging or an aging-associated disorder. In some instances, aneffective amount or dose of active agent will not only slow or halt theprogression of the disease condition but will also induce the reversalof the condition, i.e., will cause an improvement in cognitive ability.For example, in some instances, an effective amount is the amount thatwhen administered for a suitable period of time, usually at least aboutone week, and maybe about two weeks, or more, up to a period of about 3weeks, 4 weeks, 8 weeks, or longer will improve the cognitive abilitiesof an individual suffering from an aging-associated cognitive impairmentby, for example 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, in someinstances 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold or more relative tocognition prior to administration of the blood product.

Where desired, effectiveness of treatment may be assessed using anyconvenient protocol. Cognition tests and IQ test for measuring cognitiveability, e.g., attention and concentration, the ability to learn complextasks and concepts, memory, information processing, visuospatialfunction, the ability to produce and understanding language, the abilityto solve problems and make decisions, and the ability to performexecutive functions, are well known in the art, any of which may be usedto measure the cognitive ability of the individual before and/or duringand after treatment with the subject blood product, e.g., to confirmthat an effective amount has been administered. These include, forexample, the General Practitioner Assessment of Cognition (GPCOG) test,the Memory Impairment Screen, the Mini Mental State Examination (MMSE),the California Verbal Learning Test. Second Edition, Short Form, formemory, the Delis-Kaplan Executive Functioning System test, theAlzheimer's Disease Assessment Scale (ADAS-Cog), the PsychogeriatricAssessment Scale (PAS) and the like. Progression of functional brainimprovements may be detected by brain imaging techniques, such asMagnetic Resonance Imaging (MRI) or Positron Emission Tomography (PET)and the like. A wide range of additional functional assessments may beapplied to monitor activities of daily living, executive functions,mobility, etc. In some embodiments, the method comprises the step ofmeasuring cognitive ability, and detecting a decreased rate of cognitivedecline, a stabilization of cognitive ability, and/or an increase incognitive ability after administration of the blood product as comparedto the cognitive ability of the individual before the blood product wasadministered. Such measurements may be made a week or more afteradministration of the blood product, e.g., 1 week, 2 weeks, 3 weeks, ormore, for instance, 4 weeks, 6 weeks, or 8 weeks or more, e.g., 3months, 4 months, 5 months, or 6 months or more.

Biochemically, by an “effective amount” or “effective dose” of activeagent is meant an amount of active agent that will inhibit, antagonize,decrease, reduce, or suppress by about 20% or more, e.g., by 30% ormore, by 40% or more, or by 50% or more, in some instances by 60% ormore, by 70% or more, by 80% or more, or by 90% or more, in some casesby about 100%, i.e., to negligible amounts, and in some instancesreverse, the reduction in synaptic plasticity and loss of synapses thatoccurs during the natural aging process or during the progression of anaging-associated disorder. In other words, cells present in adultmammals treated in accordance with methods of the invention will becomemore responsive to cues, e.g., activity cues, which promote theformation and maintenance of synapses.

Performance of methods of the invention, e.g., as described above, maymanifest as improvements in observed synaptic plasticity, both in vitroand in vivo as an induction of long term potentiation. For example, theinduction of LTP in neural circuits may be observed in awakeindividuals, e.g., by performing non-invasive stimulation techniques onawake individuals to induce LTP-like long-lasting changes in localizedneural activity (Cooke S F, Bliss T V (2006) Plasticity in the humancentral nervous system. Brain. 129(Pt 7):1659-73); mapping plasticityand increased neural circuit activity in individuals, e.g., by usingpositron emission tomography, functional magnetic resonance imaging,and/or transcranial magnetic stimulation (Cramer and Bastings, “Mappingclinically relevant plasticity after stroke,” Neuropharmacology(2000)39:842-51); and by detecting neural plasticity following learning,i.e., improvements in memory, e.g., by assaying retrieval-related brainactivity (Buchmann et al., “Prion protein M129V polymorphism affectsretrieval-related brain activity,” Neuropsychologia. (2008) 46:2389-402)or, e.g., by imaging brain tissue by functional magnetic resonanceimaging (fMRI) following repetition priming with familiar and unfamiliarobjects (Soldan et al., “Global familiarity of visual stimuli affectsrepetition-related neural plasticity but not repetition priming,”Neuroimage. (2008) 39:515-26; Soldan at al., “Aging does not affectbrain pattems of repetition effects associated with perceptual primingof novel objects,” J. Cogn. Neurosci. (2008) 20:1762-76). In someembodiments, the method includes the step of measuring synapticplasticity, and detecting a decreased rate of loss of synapticplasticity, a stabilization of synaptic plasticity, and/or an increasein synaptic plasticity after administration of the blood product ascompared to the synaptic plasticity of the individual before the bloodproduct was administered. Such measurements may be made a week or moreafter administration of the blood product, e.g., 1 week, 2 weeks, 3weeks, or more, for instance, 4 weeks, 6 weeks, or 8 weeks or more,e.g., 3 months, 4 months, 5 months, or 6 months or more.

In some instances, the methods result in a change in expression levelsof one or more genes in one or more tissues of the host, e.g., ascompared to a suitable control (such as described in the Experimentalsection, below). The change in expression level of a given gene may be0.5 fold or greater, such as 1.0 fold or greater, including 1.5 fold orgreater. The tissue may vary, and in some instances is nervous systemtissue, e.g., central nervous system tissue, including brain tissue,e.g., hippocampal tissue. In some instances, the modulation ofhippocampal gene expression is manifested as enhanced hippocampalplasticity, e.g., as compared to a suitable control.

In some instances, treatment results in an enhancement in the levels ofone or more proteins in one or more tissues of the host, e.g., ascompared to a suitable control (such as described in the Experimentalsection, below). The change in protein level of a given protein may be0.5 fold or greater, such as 1.0 fold or greater, including 1.5 fold orgreater, where in some instances the level may approach that of ahealthy wild-type level, e.g., within 50% or less, such as 25% or less,including 10% or less, e.g., 5% or less of the healthy wild-type level.The tissue may vary, and in some instances is nervous system tissue,e.g., central nervous system tissue, including brain tissue, e.g.,hippocampal tissue.

In some instances, the methods result in one or more structural changesin one or more tissues. The tissue may vary, and in some instances isnervous system tissue, e.g., central nervous system tissue, includingbrain tissue, e.g., hippocampal tissue. Structure changes of interestinclude an increase in dendritic spine density of mature neurons in thedentate gyrus (DG) of the hippocampus, e.g., as compared to a suitablecontrol. In some instances, the modulation of hippocampal structure ismanifested as enhanced synapse formation, e.g., as compared to asuitable control. In some instances, the methods may result in anenhancement of long term potentiation, e.g., as compared to a suitablecontrol.

In some instances, practice of the methods, e.g., as described above,results in an increase in neurogenesis in the adult mammal. The increasemay be identified in a number of different ways, e.g., as describedbelow in the Experimental section. In some instances, the increase inneurogenesis manifests as an increase the amount of Dcx-positiveimmature neurons, e.g., where the increase may be 2-fold or greater. Insome instances, the increase in neurogenesis manifests as an increase inthe number of BrdU/NeuN positive cells, where the increase may be 2-foldor greater.

In some instances, the methods result in enhancement in learning andmemory, e.g., as compared to a suitable control. Enhancement in learningand memory may be evaluated in a number of different ways, e.g., thecontextual fear conditioning and/or radial arm water maze (RAWM)paradigms described in the experimental section, below. When measured bycontextual fear conditioning, treatment results in some instances inincreased freezing in contextual, but not cued, memory testing. Whenmeasured by RAWM, treatment results in some instances in enhancedlearning and memory for platform location during the testing phase ofthe task. In some instances, treatment is manifested as enhancedcognitive improvement in hippocampal-dependent learning and memory,e.g., as compared to a suitable control.

In some embodiments, B2M level reduction, e.g., as described above, maybe performed in conjunction with an active agent having activitysuitable to treat aging-associated cognitive impairment. For example, anumber of active agents have been shown to have some efficacy intreating the cognitive symptoms of Alzheimer's disease (e.g., memoryloss, confusion, and problems with thinking and reasoning), e.g.,cholinesterase inhibitors (e.g., Donepezil, Rivastigmine, Galantamine,Tacrine), Memantine, and Vitamin E. As another example, a number ofagents have been shown to have some efficacy in treating behavioral orpsychiatric symptoms of Alzheimer's Disease, e.g., citalopram (Celexa),fluoxetine (Prozac), paroxeine (Paxil), sertraline (Zoloft), trazodone(Desyrel), lorazepam (Ativan), oxazepam (Serax), aripiprazole (Abilify),clozapine (Clozaril), haloperidol (Haldol), olanzapine (Zyprexa),quetiapine (Seroquel), risperidone (Risperdal), and ziprasidone(Geodon).

In some aspects of the subject methods, the method further comprises thestep of measuring cognition and/or synaptic plasticity after treatment,e.g., using the methods described herein or known in the art, anddetermining that the rate of cognitive decline or loss of synapticplasticity have been reduced and/or that cognitive ability or synapticplasticity have improved in the individual. In some such instances, thedetermination is made by comparing the results of the cognition orsynaptic plasticity test to the results of the test performed on thesame individual at an earlier time, e.g., 2 weeks earlier, 1 monthearlier, 2 months earlier, 3 months earlier, 6 months earlier, 1 yearearlier, 2 years earlier, 5 years earlier, or 10 years earlier, or more.

In some embodiments, the subject methods further include diagnosing anindividual as having a cognitive impairment, e.g., using the methodsdescribed herein or known in the art for measuring cognition andsynaptic plasticity, prior to administering the subjectplasma-comprising blood product. In some instances, the diagnosing willcomprise measuring cognition and/or synaptic plasticity and comparingthe results of the cognition or synaptic plasticity test to one or morereferences, e.g., a positive control and/or a negative control. Forexample, the reference may be the results of the test performed by oneor more age-matched individuals that experience aging-associatedcognitive impairments (i.e., positive controls) or that do notexperience aging-associated cognitive impairments (i.e., negativecontrols). As another example, the reference may be the results of thetest performed by the same individual at an earlier time, e.g., 2 weeksearlier, 1 month earlier, 2 months earlier, 3 months earlier, 6 monthsearlier, 1 year earlier, 2 years earlier, 5 years earlier, or 10 yearsearlier, or more.

In some embodiments, the subject methods further comprise diagnosing anindividual as having an aging-associated disorder, e.g., Alzheimer'sdisease, Parkinson's disease, frontotemporal dementia, progressivesupranuclear palsy, Huntington's disease, amyotrophic lateral sclerosis,spinal muscular atrophy, multiple sclerosis, multi-system atrophy,glaucoma, ataxias, myotonic dystrophy, dementia, and the like. Methodsfor diagnosing such aging-associated disorders are well-known in theart, any of which may be used by the ordinarily skilled artisan indiagnosing the individual. In some embodiments, the subject methodsfurther comprise both diagnosing an individual as having anaging-associated disorder and as having a cognitive impairment.

Utility

The subject methods find use in treating, including preventing,aging-associated impairments and conditions associated therewith, suchas impairments in the cognitive ability of individuals. Individualssuffering from or at risk of developing an aging-associated cognitiveimpairments include individuals that are about 50 years old or older,e.g., 60 years old or older, 70 years old or older, 80 years old orolder, 90 years old or older, and usually no older than 100 years old,i.e., between the ages of about 50 and 100, e.g., 50, 55, 60, 65, 70,75, 80, 85, 90, 95 or about 100 years old, and are suffering fromcognitive impairment associated with natural aging process, e.g., mildcognitive impairment (M.C.I.); and individuals that are about 50 yearsold or older, e.g., 60 years old or older, 70 years old or older, 80years old or older, 90 years old or older, and usually no older than 100years old, i.e., between the ages of about 50 and 90, e.g., 50, 55, 60,65, 70, 75, 80, 85, 90, 95 or about 100 years old, that have not yetbegun to show symptoms of cognitive impairment. Examples of cognitiveimpairments that are due to natural aging include the following:

Mild cognitive impairment (M.C.I.) is a modest disruption of cognitionthat manifests as problems with memory or other mental functions such asplanning, following instructions, or making decisions that have worsenedover time while overall mental function and daily activities are notimpaired. Thus, although significant neuronal death does not typicallyoccur, neurons in the aging brain are vulnerable to sub-lethalage-related alterations in structure, synaptic integrity, and molecularprocessing at the synapse, all of which impair cognitive function.

Individuals suffering from or at risk of developing an aging-associatedcognitive impairment that will benefit from treatment with the subjectplasma-comprising blood product, e.g., by the methods disclosed herein,also include individuals of any age that are suffering from a cognitiveimpairment due to an aging-associated disorder; and individuals of anyage that have been diagnosed with an aging-associated disorder that istypically accompanied by cognitive impairment, where the individual hasnot yet begun to present with symptoms of cognitive impairment. Examplesof such aging-associated disorders include the following:

Alzheimer's disease (AD). Alzheimer's disease is a progressive,inexorable loss of cognitive function associated with an excessivenumber of senile plaques in the cerebral cortex and subcortical graymatter, which also contains b-amyloid and neurofibrillary tanglesconsisting of tau protein. The common form affects persons >60 yr old,and its incidence increases as age advances. It accounts for more than65% of the dementias in the elderly.

The cause of Alzheimer's disease is not known. The disease runs infamilies in about 15 to 20% of cases. The remaining, so-called sporadiccases have some genetic determinants. The disease has an autosomaldominant genetic pattern in most early-onset and some late-onset casesbut a variable late-life penetrance. Environmental factors are the focusof active investigation.

In the course of the disease, synapses, and ultimately neurons are lostwithin the cerebral cortex, hippocampus, and subcortical structures(including selective cell loss in the nucleus basalis of Meynert), locuscaeruleus, and nucleus raphae dorsalis. Cerebral glucose use andperfusion is reduced in some areas of the brain (parietal lobe andtemporal cortices in early-stage disease, prefrontal cortex inlate-stage disease). Neuritic or senile plaques (composed of neurites,astrocytes, and glial cells around an amyloid core) and neurofibrillarytangles (composed of paired helical filaments) play a role in thepathogenesis of Alzheimer's disease. Senile plaques and neurofibrillarytangles occur with normal aging, but they are much more prevalent inpersons with Alzheimer's disease.

Parkinson's Disease. Parkinson's Disease (PD) is an idiopathic, slowlyprogressive, degenerative CNS disorder characterized by slow anddecreased movement, muscular rigidity, resting tremor, and posturalinstability. Originally considered primarily a motor disorder, PD is nowrecognized to also affect cognition, behavior, sleep, autonomicfunction, and sensory function. The most common cognitive impairmentsinclude an impairment in attention and concentration, working memory,executive function, producing language, and visuospatial function.

In primary Parkinson's disease, the pigmented neurons of the substantianigra, locus caeruleus, and other brain stem dopaminergic cell groupsare lost. The cause is not known. The loss of substantia nigra neurons,which project to the caudate nucleus and putamen, results in depletionof the neurotransmitter dopamine in these areas. Onset is generallyafter age 40, with increasing incidence in older age groups.

Secondary parkinsonism results from loss of or interference with theaction of dopamine in the basal ganglia due to other idiopathicdegenerative diseases, drugs, or exogenous toxins. The most common causeof secondary parkinsonism is ingestion of antipsychotic drugs orreserpine, which produce parkinsonism by blocking dopamine receptors.Less common causes include carbon monoxide or manganese poisoning,hydrocephalus, structural lesions (tumors, infarcts affecting themidbrain or basal ganglia), subdural hematoma, and degenerativedisorders, including striatonigral degeneration.

Frontotemporal dementia. Frontotemporal dementia (FTD) is a conditionresulting from the progressive deterioration of the frontal lobe of thebrain. Over time, the degeneration may advance to the temporal lobe.Second only to Alzheimer's disease (AD) in prevalence, FTD accounts for20% of pre-senile dementia cases. Symptoms are classified into threegroups based on the functions of the frontal and temporal lobesaffected: Behavioural variant FTD (bvFTD), with symptoms includelethargy and aspontaneity on the one hand, and disinhibition on theother; progressive nonfluent aphasia (PNFA), in which a breakdown inspeech fluency due to articulation difficulty, phonological and/orsyntactic errors is observed but word comprehension is preserved; andsemantic dementia (SD), in which patients remain fluent with normalphonology and syntax but have increasing difficulty with naming and wordcomprehension. Other cognitive symptoms common to all FTD patientsinclude an impairment in executive function and ability to focus. Othercognitive abilities, including perception, spatial skills, memory andpraxis typically remain intact. FTD can be diagnosed by observation ofreveal frontal lobe and/or anterior temporal lobe atrophy in structuralMRI scans.

A number of forms of FTD exist, any of which may be treated or preventedusing the subject methods and compositions. For example, one form offrontotemporal dementia is Semantic Dementia (SD). SD is characterizedby a loss of semantic memory in both the verbal and non-verbal domains.SD patients often present with the complaint of word-findingdifficulties. Clinical signs include fluent aphasia, anomia, impairedcomprehension of word meaning, and associative visual agnosia (theinability to match semantically related pictures or objects). As thedisease progresses, behavioral and personality changes are often seensimilar to those seen in frontotemporal dementia although cases havebeen described of ‘pure’ semantic dementia with few late behavioralsymptoms. Structural MRI imaging shows a characteristic pattern ofatrophy in the temporal lobes (predominantly on the left), with inferiorgreater than superior involvement and anterior temporal lobe atrophygreater than posterior.

As another example, another form of frontotemporal dementia is Pick'sdisease (PiD, also PcD). A defining characteristic of the disease isbuild-up of tau proteins in neurons, accumulating into silver-staining,spherical aggregations known as “Pick bodies”. Symptoms include loss ofspeech (aphasia) and dementia. Patients with orbitofrontal dysfunctioncan become aggressive and socially inappropriate. They may steal ordemonstrate obsessive or repetitive stereotyped behaviors. Patients withdorsomedial or dorsolateral frontal dysfunction may demonstrate a lackof concern, apathy, or decreased spontaneity. Patients can demonstratean absence of self-monitoring, abnormal self-awareness, and an inabilityto appreciate meaning. Patients with gray matter loss in the bilateralposterolateral orbitofrontal cortex and right anterior insula maydemonstrate changes in eating behaviors, such as a pathologic sweettooth. Patients with more focal gray matter loss in the anterolateralorbitofrontal cortex may develop hyperphagia. While some of the symptomscan initially be alleviated, the disease progresses and patients oftendie within two to ten years.

Huntington's disease. Huntington's disease (HD) is a hereditaryprogressive neurodegenerative disorder characterized by the developmentof emotional, behavioral, and psychiatric abnormalities; loss ofintellectual or cognitive functioning; and movement abnormalities (motordisturbances). The classic signs of HD include the development ofchorea—involuntary, rapid, irregular, jerky movements that may affectthe face, arms, legs, or trunk—as well as cognitive decline includingthe gradual loss of thought processing and acquired intellectualabilities. There may be impairment of memory, abstract thinking, andjudgment; improper perceptions of time, place, or identity(disorientation); increased agitation; and personality changes(personality disintegration). Although symptoms typically become evidentduring the fourth or fifth decades of life, the age at onset is variableand ranges from early childhood to late adulthood (e.g., 70s or 80s).

HD is transmitted within families as an autosomal dominant trait. Thedisorder occurs as the result of abnormally long sequences or “repeats”of coded instructions within a gene on chromosome 4 (4p16.3). Theprogressive loss of nervous system function associated with HD resultsfrom loss of neurons in certain areas of the brain, including the basalganglia and cerebral cortex.

Amyotrophic lateral sclerosis. Amyotrophic lateral sclerosis (ALS) is arapidly progressive, invariably fatal neurological disease that attacksmotor neurons. Muscular weakness and atrophy and signs of anterior horncell dysfunction are initially noted most often in the hands and lessoften in the feet. The site of onset is random, and progression isasymmetric. Cramps are common and may precede weakness. Rarely, apatient survives 30 years; 50% die within 3 years of onset. 20% live 5years, and 10% live 10 years. Diagnostic features include onset duringmiddle or late adult life and progressive, generalized motor involvementwithout sensory abnormalities. Nerve conduction velocities are normaluntil late in the disease. Recent studies have documented thepresentation of cognitive impairments as well, particularly a reductionin immediate verbal memory, visual memory, language, and executivefunction.

A decrease in cell body area, number of synapses and total synapticlength has been reported in even normal-appearing neurons of the ALSpatients. It has been suggested that when the plasticity of the activezone reaches its limit, a continuing loss of synapses can lead tofunctional impairment. Promoting the formation or new synapses orpreventing synapse loss may maintain neuron function in these patients.

Multiple Sclerosis. Multiple Sclerosis (MS) is characterized by varioussymptoms and signs of CNS dysfunction, with remissions and recurringexacerbations. The most common presenting symptoms are parenthesis inone or more extremities, in the trunk, or on one side of the face;weakness or clumsiness of a leg or hand; or visual disturbances, e.g.,partial blindness and pain in one eye (retrobulbar optic neuritis),dimness of vision, or scotomas. Common cognitive impairments includeimpairments in memory (acquiring, retaining, and retrieving newinformation), attention and concentration (particularly dividedattention), information processing, executive functions, visuospatialfunctions, and verbal fluency. Common early symptoms are ocular palsyresulting in double vision (diplopia), transient weakness of one or moreextremities, slight stiffness or unusual fatigability of a limb, minorgait disturbances, difficulty with bladder control, vertigo, and mildemotional disturbances; all indicate scattered CNS involvement and oftenoccur months or years before the disease is recognized. Excess heat mayaccentuate symptoms and signs.

The course is highly varied, unpredictable, and, in most patients,remittent. At first, months or years of remission may separate episodes,especially when the disease begins with retrobulbar optic neuritis.However, some patients have frequent attacks and are rapidlyincapacitated; for a few the course can be rapidly progressive.

Glaucoma. Glaucoma is a common neurodegenerative disease that affectsretinal ganglion cells (RGCs). Evidence supports the existence ofcompartmentalized degeneration programs in synapses and dendrites,including in RGCs. Recent evidence also indicates a correlation betweencognitive impairment in older adults and glaucoma (Yochim B P, et al.Prevalence of cognitive impairment, depression, and anxiety symptomsamong older adults with glaucoma. J Glaucoma. 2012; 21(4):250-254).

Myotonic dystrophy. Myotonic dystrophy (DM) is an autosomal dominantmultisystem disorder characterized by dystrophic muscle weakness andmyotonia. The molecular defect is an expanded trinucleotide (CTG) repeatin the 3′ untranslated region of the myotonin-protein kinase gene onchromosome 19q. Symptoms can occur at any age, and the range of clinicalseverity is broad. Myotonia is prominent in the hand muscles, and ptosisis common even in mild cases. In severe cases, marked peripheralmuscular weakness occurs, often with cataracts, premature balding,hatchet facies, cardiac arrhythmias, testicular atrophy, and endocrineabnormalities (e.g., diabetes mellitus). Mental retardation is common insevere congenital forms, while an aging-related decline of frontal andtemporal cognitive functions, particularly language and executivefunctions, is observed in milder adult forms of the disorder. Severelyaffected persons die by their early 50s.

Dementia. Dementia describes class of disorders having symptomsaffecting thinking and social abilities severely enough to interferewith daily functioning. Other instances of dementia in addition to thedementia observed in later stages of the aging-associated disordersdiscussed above include vascular dementia, and dementia with Lewybodies, described below.

In vascular dementia, or “multi-infarct dementia”, cognitive impairmentis caused by problems in supply of blood to the brain, typically by aseries of minor strokes, or sometimes, one large stroke preceded orfollowed by other smaller strokes. Vascular lesions can be the result ofdiffuse cerebrovascular disease, such as small vessel disease, or focallesions, or both. Patients suffering from vascular dementia present withcognitive impairment, acutely or subacutely, after an acutecerebrovascular event, after which progressive cognitive decline isobserved. Cognitive impairments are similar to those observed inAlzheimer's disease, including impairments in language, memory, complexvisual processing, or executive function, although the related changesin the brain are not due to AD pathology but to chronic reduced bloodflow in the brain, eventually resulting in dementia. Single photonemission computed tomography (SPECT) and positron emission tomography(PET) neuroimaging may be used to confirm a diagnosis of multi-infarctdementia in conjunction with evaluations involving mental statusexamination.

Dementia with Lewy bodies (DLB, also known under a variety of othernames including Lewy body dementia, diffuse Lewy body disease, corticalLewy body disease, and senile dementia of Lewy type) is a type ofdementia characterized anatomically by the presence of Lewy bodies(clumps of alpha-synuclein and ubiquitin protein) in neurons, detectablein post mortem brain histology. Its primary feature is cognitivedecline, particularly of executive functioning. Alertness and short termmemory will rise and fall. Persistent or recurring visual hallucinationswith vivid and detailed pictures are often an early diagnostic symptom.DLB it is often confused in its early stages with Alzheimer's diseaseand/or vascular dementia, although, where Alzheimer's disease usuallybegins quite gradually, DLB often has a rapid or acute onset. DLBsymptoms also include motor symptoms similar to those of Parkinson's.DLB is distinguished from the dementia that sometimes occurs inParkinson's disease by the time frame in which dementia symptoms appearrelative to Parkinson symptoms. Parkinson's disease with dementia (PDD)would be the diagnosis when dementia onset is more than a year after theonset of Parkinson's. DLB is diagnosed when cognitive symptoms begin atthe same time or within a year of Parkinson symptoms.

Progressive supranuclear palsy. Progressive supranuclear palsy (PSP) isa brain disorder that causes serious and progressive problems withcontrol of gait and balance, along with complex eye movement andthinking problems. One of the classic signs of the disease is aninability to aim the eyes properly, which occurs because of lesions inthe area of the brain that coordinates eye movements. Some individualsdescribe this effect as a blurring. Affected individuals often showalterations of mood and behavior, including depression and apathy aswell as progressive mild dementia. The disorder's long name indicatesthat the disease begins slowly and continues to get worse (progressive),and causes weakness (palsy) by damaging certain parts of the brain abovepea-sized structures called nuclei that control eye movements(supranuclear). PSP was first described as a distinct disorder in 1964,when three scientists published a paper that distinguished the conditionfrom Parkinson's disease. It is sometimes referred to asSteele-Richardson-Olszewski syndrome, reflecting the combined names ofthe scientists who defined the disorder. Although PSP gets progressivelyworse, no one dies from PSP itself.

Ataxia. People with ataxia have problems with coordination because partsof the nervous system that control movement and balance are affected.Ataxia may affect the fingers, hands, arms, legs, body, speech, and eyemovements. The word ataxia is often used to describe a symptom ofincoordination which can be associated with infections, injuries, otherdiseases, or degenerative changes in the central nervous system. Ataxiais also used to denote a group of specific degenerative diseases of thenervous system called the hereditary and sporadic ataxias which are theNational Ataxia Foundation's primary emphases.

Multiple-system atrophy. Multiple-system atrophy (MSA) is a degenerativeneurological disorder. MSA is associated with the degeneration of nervecells in specific areas of the brain. This cell degeneration causesproblems with movement, balance, and other autonomic functions of thebody such as bladder control or blood-pressure regulation. The cause ofMSA is unknown and no specific risk factors have been identified. Around55% of cases occur in men, with typical age of onset in the late 50s toearly 60s. MSA often presents with some of the same symptoms asParkinson's disease. However, MSA patients generally show minimal if anyresponse to the dopamine medications used for Parkinson's.

In some embodiments, the subject methods and compositions find use inslowing the progression of aging-associated cognitive impairment. Inother words, cognitive abilities in the individual will decline moreslowly following treatment by the disclosed methods than prior to or inthe absence of treatment by the disclosed methods. In some suchinstances, the subject methods of treatment include measuring theprogression of cognitive decline after treatment, and determining thatthe progression of cognitive decline is reduced. In some such instances,the determination is made by comparing to a reference, e.g., the rate ofcognitive decline in the individual prior to treatment, e.g., asdetermined by measuring cognition prior at two or more time points priorto administration of the subject blood product.

The subject methods and compositions also find use in stabilizing thecognitive abilities of an individual, e.g., an individual suffering fromaging-associated cognitive decline or an individual at risk of sufferingfrom aging-associated cognitive decline. For example, the individual maydemonstrate some aging-associated cognitive impairment, and progressionof cognitive impairment observed prior to treatment with the disclosedmethods will be halted following treatment by the disclosed methods. Asanother example, the individual may be at risk for developing anaging-associated cognitive decline (e.g., the individual may be aged 50years old or older, or may have been diagnosed with an aging-associateddisorder), and the cognitive abilities of the individual aresubstantially unchanged, i.e., no cognitive decline can be detected,following treatment by the disclosed methods as compared to prior totreatment with the disclosed methods.

The subject methods and compositions also find use in reducing cognitiveimpairment in an individual suffering from an aging-associated cognitiveimpairment. In other words, cognitive ability is improved in theindividual following treatment by the subject methods. For example, thecognitive ability in the individual is increased, e.g., by 2-fold ormore, 5-fold or more, 10-fold or more, 15-fold or more, 20-fold or more,30-fold or more, or 40-fold or more, including 50-fold or more, 60-foldor more, 70-fold or more, 80-fold or more, 90-fold or more, or 100-foldor more, following treatment by the subject methods relative to thecognitive ability that is observed in the individual prior to treatmentby the subject methods. In some instances, treatment by the subjectmethods and compositions restores the cognitive ability in theindividual suffering from aging-associated cognitive decline, e.g., totheir level when the individual was about 40 years old or less. In otherwords, cognitive impairment is abrogated.

Reagents, Devices and Kits

Also provided are reagents, devices and kits thereof for practicing oneor more of the above-described methods. The subject reagents, devicesand kits thereof may vary greatly. Reagents and devices of interestinclude those mentioned above with respect to the methods of reducingB2M levels in an adult mammal.

In addition to the above components, the subject kits will furtherinclude instructions for practicing the subject methods. Theseinstructions may be present in the subject kits in a variety of forms,one or more of which may be present in the kit. One form in which theseinstructions may be present is as printed information on a suitablemedium or substrate, e.g., a piece or pieces of paper on which theinformation is printed, in the packaging of the kit, in a packageinsert, etc. Yet another means would be a computer readable medium,e.g., diskette, CD, portable flash drive, etc., on which the informationhas been recorded. Yet another means that may be present is a websiteaddress which may be used via the internet to access the information ata removed site. Any convenient means may be present in the kits.

The following examples are provided by way of illustration and not byway of limitation.

EXPERIMENTAL

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g., amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Centigrade,and pressure is at or near atmospheric.

I. Methods A. Animal Models.

The following mouse lines were used: C57BL/6 (The Jackson Laboratory),C57BL/6 aged mice (National Institutes of Aging), β2-Microglobulin(B2M−/−) mutant mice and transporter associated with antigen processing1 (Tap1−/−) mutant mice (The Jackson Laboratory). All studies were donein male mice. The numbers of mice used to result in statisticallysignificant differences was calculated using standard power calculationswith α=0.05 and a power of 0.8. We used an online tool(http://www.stat.uiowa.edu/˜rlenth/Power/index.html) to calculate powerand sample size based on experience with the respective tests,variability of the assays and inter-individual differences withingroups. Mice were housed under specific pathogen-free conditions under a12 h light-dark cycle and all animal handling and use was in accordancewith institutional guidelines approved by the University of CaliforniaSan Francisco IACUC and the VA Palo Alto Committee on Animal Research.

B. Parabiosis.

Parabiosis surgery followed previously described procedures (Villeda, S.A., et al., “The ageing systemic milieu negatively regulatesneurogenesis and cognitive function.,” Nature (2011) 477: 90-94),(Villeda, S. A. et al., “Young blood reverses age-related impairments incognitive function and synaptic plasticity in mice.,” Nature medicine(2014) 20:659-663). Mirror-image incisions at the left and right flankswere made through the skin and shorter incisions made through theabdominal wall. The peritoneal openings of the adjacent parabionts weresutured together. Elbow and knee joints from each parabiont were suturedtogether and the skin of each mouse was stapled (9 mm Autoclip, ClayAdams) to the skin of the adjacent parabiont. Each mouse was injectedsubcutaneously with Baytril antibiotic and Buprenex as directed for painand monitored during recovery. For overall health and maintenancebehavior, several recovery characteristics were analyzed at varioustimes after surgery, including paired weights and grooming behavior.

C. Stereotaxic Injections.

Animals were placed in a stereotaxic frame and anesthetized with 2%isoflurane (2 L/min oxygen flow rate) delivered through an anesthesianose cone. Ophthalmic eye ointment (Puralube Vet Ointment, Dechra) wasapplied to the cornea to prevent desiccation during surgery. The areaaround the incision was trimmed. Solutions were injected bilaterallyinto the DG of the dorsal hippocampi using the following coordinates:(from bregma) anterior=−2 mm, lateral=1.5 mm, (from skull surface)height=−2.1 mm. A 2 μl volume was injected stereotaxically over 10minutes (injection speed: 0.20 μl/min) using a 5 μl 26s gauge Hamiltonsyringe. To limit reflux along the injection track, the needle wasmaintained in situ for 8 minutes, slowly pulled out half way and kept inposition for an additional two minutes. The skin was closed using silksuture. Each mouse was injected subcutaneously with the analgesicBuprenex. Mice were single-housed and monitored during recovery.

D. B2M Administration.

Carrier free purified human β2-Microglobulin (Lee Biosolutions) wasdissolved in PBS and administered systemically (100 μg/kg) viaintraorbital in young (3 months) wild type animals, or stereotaxically(0.50 μl; 0.1 μg/μl) into the DG of the hippocampus in young (3 months)wild type and Tap1^(−/−) mutant. For histological analysis B2M andvehicle were administered into contralateral DG of the same animal. Forbehavioral analysis B2M or vehicle were administered bilaterally intothe DG and mice were allowed to recover for six or 30 days prior tocognitive testing.

E. BrdU Administration and Quantification.

For short term Brdu labeling 50 mg/kg of BrdU was injectedintraperitoneally into mice daily either three or six days beforesacrifice. For long term BrdU labeling 50 mg/kg of BrdU was injectedinto mice once a day for six days and animals were sacrificed 28 daysafter first administration. To estimate the total number ofBrdU-positive cells in the brain, we performed DAB staining for BrdU onevery sixth hemibrain section for a total of six sections. The number ofBrdU-positive cells in the granule cell and subgranular cell layer ofthe DG were counted and multiplied by 12 to estimate the total number ofBrdU-positive cells in the entire DG. To determine the fate of dividingcells a total of 200 BrdU-positive cells across 4-6 sections per mousewere analyzed by confocal microscopy for co-expression with NeuN andGFAP. The number of double-positive cells was expressed as a percentageof BrdU-positive cells.

F. Immunohistochemistry.

Tissue processing and immunohistochemistry was performed onfree-floating sections following standard published techniques (Villeda,S. A., et al., “The ageing systemic milieu negatively regulatesneurogenesis and cognitive function.,” Nature (2011) 477: 90-94).Briefly, mice were anesthetized with 400 mg/kg chloral hydrate(Sigma-Aldrich) and transcardially perfused with 0.9% saline. Brainswere removed and fixed in phosphate-buffered 4% paraformaldehyde, pH7.4, at 4° C. for 48 h before they were sunk through 30% sucrose forcryoprotection. Brains were then sectioned coronally at 40 in with acryomicrotome (Leica Camera, Inc.) and stored in cryoprotective medium.Primary antibodies were: goat anti-Dcx (1:500; Santa Cruz Biotechnology;sc-8066, clone: C-18), rat anti-BrdU (1:5000, Accurate Chemical andScientific Corp.; ab6326, clone: BU1/75), mouse anti-Nestin (1:500;Millipore; MAB353; clone: rat-401) MCM2 (1:500, BD Biosciences; 610700;clone: 46/BM28), chicken anti-Tbr2 (1:500; Millipore; AB15894), mouseanti-NeuN (1:1000; Millipore; MAB377; clone: A60), rabbit anti-GFAP(1:500; DAKO; Z0334). After overnight incubation, primary antibodystaining was revealed using biotinylated secondary antibodies (Vector)and the ABC kit (Vector) with Diaminobenzidine (DAB, Sigma-Aldrich) orfluorescence conjugated secondary antibodies (Life Technologies). ForBrdU labeling, brain sections were pre-treated with 2N HCl at 37° C. for30 min and washed three times with Tris-Buffered Saline with Tween(TBST) before incubation with primary antibody. For Nestin and Tbr2labeling, brain sections were pre-treated three times with 0.1M Citrateat 95° C. for 5 min and washed three times with Tris-Buffered Salinewith Tween (TBST) before incubation with primary antibody. To estimatethe total number of Dcx positive cells per DG immunopositive cells inthe granule cell and subgranular cell layer of the DG were counted inevery sixth coronal hemibrain section through the hippocampus for atotal of six sections and multiplied by 12.

G. Western Blot Analysis.

Mouse hippocampi were dissected after perfusion of animals, snap frozenand lysed in RIPA lysis buffer (500 mM Tris, pH 7.4, 150 mM NaCl, 0.5%Na deoxycholate, 1% NP40, 0.1% SDS, and complete protease inhibitors;Roche). Tissue lysates were mixed with 4× NuPage LDS loading buffer(Invitrogen) and loaded on a 4-12% SDS polyacrylamide gradient gel(Invitrogen) and subsequently transferred onto a nitrocellulosemembrane. The blots were blocked in 5% milk in Tris-Buffered Saline withTween (TBST) and incubated with rabbit anti-actin (1:5000, Sigma; A5060)and rabbit anti-B2M (1:2500, Abcam; ab75853; clone: EP2978Y).Horseradish peroxidase-conjugated secondary antibodies (1:5000, GEHealthcare; NA934) and an ECL kit (GE Healthcare/Amersham PharmaciaBiotech) were used to detect protein signals. Multiple exposures weretaken to select images within the dynamic range of the film (GEHealthcare Amersham Hyperfilm™ ECL). Selected films were scanned (300dpi) and quantified using ImageJ software (Version 1.46k). Actin bandswere used for normalization.

H. Cell Culture Assays.

Mouse neural progenitor cells were isolated from C57BL/6 mice orDcx-reporter mice (Couillard-Despres S, et al. “In vivo optical imagingof neurogenesis: watching new neurons in the intact brain.” Molecularimaging. 2008; 7:28-34.) as previously described (Villeda, S. A., etal., “The ageing systemic milieu negatively regulates neurogenesis andcognitive function.,” Nature (2011) 477: 90-94), (Mosher K I, et al.“Neural progenitor cells regulate microglia functions and activity.”Nature neuroscience. 2012; 15:1485-1487). Brains from postnatal animals(1 day-old) were dissected to remove olfactory bulbs, cortex, cerebellumand brainstem. After removing superficial blood vessels hippocampi werefinally minced with a scalpel, digested for 30 minutes at 37° C. inDMEMB media containing 2.5 U/ml Papain (Worthington Biochemicals), 1U/ml Dispase II (Boeringher Mannheim), and 250 U/ml DNase I (WorthingtonBiochemicals) and mechanically dissociated. NSC/progenitors werepurified using a 65% Percoll gradient and plated on uncoated tissueculture dishes at a density of 10⁵ cells/cm². NPCs were cultured understandard conditions for 48 hours in NeuroBasal A medium supplementedwith penicillin (100 U/ml), streptomycin (100 mg/ml), 2 mM L-glutamine,serum-free B27 supplement without vitamin A (Sigma-Aldrich), bFGF (20ng/ml) and EGF (20 ng/ml). Carrier free forms of human recombinant B2M(Vendor) were dissolved in PBS and added to cell cultures underself-renewal conditions every other day following cell plating. Forproliferation BrdU incorporation was measured using a cell proliferationassay system that uses a peroxidase-coupled anti-BrdU antibody togetherwith a color substrate for detection (Fisher). For bioluminescenceassays Dcx-luciferase activity was measured using a luciferase assaysystem (Promega). Differentiation was assessed by immunocytochemistryusing mouse anti-MAP2 (1:1000, Sigma; M9942; clone: HM-2) and rabbitanti-GFAP (1:500, DAKO; Z0334) antibodies. Cytotoxicity was measured bylactate dehydrogenase (LDH) detection using a Pierce LDH CytotoxicityAssay system (Life Technologies).

I. Contextual Fear Conditioning.

In this task, mice learned to associate the environmental context (fearconditioning chamber) with an aversive stimulus (mild foot shock;unconditioned stimulus, US) enabling testing for hippocampal-dependentcontextual fear conditioning. As contextual fear conditioning ishippocampus and amygdala dependent, the mild foot shock was paired witha light and tone cue (conditioned stimulus, CS) in order to also assessamygdala-dependent cued fear conditioning. Conditioned fear wasdisplayed as freezing behavior. Specific training parameters are asfollows: tone duration is 30 seconds; level is 70 dB, 2 kHz; shockduration is 2 seconds; intensity is 0.6 mA. This intensity is notpainful and can easily be tolerated but will generate an unpleasantfeeling. More specifically, on day 1 each mouse was placed in afear-conditioning chamber and allowed to explore for 2 minutes beforedelivery of a 30-second tone (70 dB) ending with a 2-second foot shock(0.6 mA). Two minutes later, a second CS-US pair was delivered. On day 2each mouse was first placed in the fear-conditioning chamber containingthe same exact context, but with no CS or foot shock. Freezing wasanalyzed for 1-3 minutes. One hour later, the mice were placed in a newcontext containing a different odor, cleaning solution, floor texture,chamber walls and shape. Animals were allowed to explore for 2 minutesbefore being re-exposed to the CS. Freezing was analyzed for 1-3minutes. Freezing was measured using a FreezeScan video tracking systemand software (Cleversys, Inc).

J. Radial Arm Water Maze.

Spatial learning and memory was assessed using the radial arm water maze(RAWM) paradigm following the protocol described by Alamed et al. Nat.Protocols (2006) 1: 1671-1679). In this task the goal arm locationcontaining a platform remains constant throughout the training andtesting phase, while the start arm is changed during each trial. On dayone during the training phase, mice are trained for 15 trails, withtrials alternating between a visible and hidden platform. On day twoduring the testing phase, mice are tested for 15 trials with a hiddenplatform. Entry into an incorrect arm is scored as an error, and errorsare averaged over training blocks (three consecutive trials).Investigators were blinded to genotype and treatment when scoring.

K. Plasma Collection and Proteomic Analysis.

Mouse blood was collected into EDTA coated tubes via tail vein bleed,mandibular vein bleed, or intracardial bleed at time of sacrifice. EDTAplasma was generated by centrifugation at 1000 g of freshly collectedblood and aliquots were stored at −80° C. until use. Human plasma andCSF samples were obtained from University of Washington School ofMedicine, Veterans Affairs Northwest Network Mental Illness Research,Education, and Clinical Center, Oregon Health Science University andUniversity of California San Diego. Subjects were chosen based onstandardized inclusion and exclusion criteria as previously described(Villeda, S. A., et al., “The ageing systemic milieu negativelyregulates neurogenesis and cognitive function.,” Nature (2011) 477:90-94), (Zhang, J. et al., “CSF multianalyte profile distinguishesAlzheimer and Parkinson diseases.,” American journal of clinicalpathology (2008) 129: 526-529), (Li, G. et al., “Cerebrospinal fluidconcentration of brain-derived neurotrophic factor and cognitivefunction in non-demented subjects.,” PloS one (2009) 4: e5424) andoutlined in Supplementary Table 1. Informed consent was obtained fromhuman subjects according to the institutional review board guidelines atthe respective centers.

TABLE 1 Normal aging subject inclusion criteria Normal aging subjectexclusion criteria Age: Subject meets age cutoffs for Vision and/orhearing too impaired (even with entry to the specific diagnostic group.correction) to allow compliance with Informant: Presence of an informantpsychometric testing for all subjects. Medical problems: unstable,poorly controlled, General health: good enough to or severe medicalproblems or diseases. complete study visits. Cancer in the past 12months (excludes Body Mass Index (BMI): 18-34 squamous CA of the skin orstage 1 prostate CA). Stable medications for 4 weeks beforeContraindications to lumbar puncture: Bleeding the visit to draw bloodor CSF. disorder, use of Coumadin, heparin or similar Permittedmedications include: AChE- anticoagulant, platelets < 100,000; deformityinhibitors, Memantine, HRT or surgery affecting lumbosacral spine which(estrogen +/− progesterone, Lupron), is severe enough to make lumbarpuncture Thyroid hormone, difficult, cutaneous sepsis at lumbosacralAntidepressants, statins. region. Normal basic laboratory tests: BUN,Neurological disorders: neurodegenerative creatinine (will allowcreatinine up diseases such as Alzheimer's Disease, Parkinson's to 1.5),B12, TSH. Disease, CJD, FTD, PSP; stroke in past 12 months MMSE > 27/30(exemptions if low or severe enough residual effects of earlier strokeeducation and control status to impair neurological or cognitivefunction; established by detailed evaluation) Multiple sclerosis;epilepsy Memory performance on logical Psychiatric disorders:schizophrenia, bipolar Memory within normal limits. affective disorderCDR = 0 Active/uncontrolled depression: by history or Neurological examis normal, i.e. no GDS score evidence of stroke, Parkinsonism Drug oralcohol abuse in past 2 years or major abnormalities. Exclusionarymedications (in 4 weeks before visit to draw blood or CSF)Neuroleptics/atypical antipsychotics Anti-Parkinson's Diseasemedications (L-dopa, dopamine agonists) CNS stimulants: modafinil,Ritalin Antiepileptic drugs (exceptions for Neurontin or similar newerAEDs given for pain control) Insulin treatment Cortisone (oralprohibited-topical or inhaler use allowed), anti-immune drugs (e.g.methotrexate, cytoxan, IVIg, tacrolimus, cyclosporine) or antineoplasticdrugs Anti-HIV medications

The plasma concentrations of cytokines and signaling molecules weremeasured in human and mouse plasma samples using standard antibody-basedmultiplex immunoassays (Luminex) by Rules Based Medicine Inc., afee-for-service provider. All Luminex measurements where obtained in ablinded fashion. All assays were developed and validated to ClinicalLaboratory Standards Institute (formerly NCCLS) guidelines based uponthe principles of immunoassay as described by the manufacturers.

L. Data and Statistical Analysis.

All experiments were randomized and blinded by an independent researcherprior to pharmacological treatment or assessment of genetic mousemodels. Researchers remained blinded throughout histological,biochemical and behavioral assessments. Groups were un-blinded at theend of each experiment upon statistical analysis. Data are expressed asmean±SEM. The distribution of data in each set of experiments was testedfor normality using D'Agostino-Pearson omnibus test or Shapiro-Wilktest. No significant differences in variance between groups weredetected using an F test. Statistical analysis was performed with Prism5.0 software (GraphPad Software). Means between two groups were comparedwith two-tailed, unpaired Student's t test. Comparisons of means frommultiple groups with each other or against one control group wereanalyzed with 1-way ANOVA followed by appropriate post-hoc tests(indicated in figure legends).

II. Results and Discussion

Aging remains the single most dominant risk factor for dementia-relatedneurodegenerative diseases, such as Alzheimer's disease (Hedden &Gabrieli, “Insights into the ageing mind: a view from cognitiveneuroscience.,” Nature reviews. Neuroscience (2004) 5:87-96; Mattson &Magnus, “Ageing and neuronal vulnerability.,” Nature reviews.Neuroscience (2006) 7:278-294; Small et al., “A pathophysiologicalframework of hippocampal dysfunction in ageing and disease.,” Naturereviews. Neuroscience (2011) 12:585-601). As such, it is imperative togain mechanistic insight into what drives aging phenotypes in the brainin order to maintain cognitive integrity in the elderly, andconsequently counteract vulnerability to neurodegenerative disease. We,and others, have recently shown that systemic manipulations such asheterochronic parabiosis (in which the circulatory system of a young andold animal are joined) or young plasma administration can partiallyreverse age-related loss of cognitive and regenerative faculties in theaged brain (Katsimpardi et al., “Vascular and neurogenic rejuvenation ofthe aging mouse brain by young systemic factors.,” Science (2014)344:630-634; Villeda et al., “The ageing systemic milieu negativelyregulates neurogenesis and cognitive function.,” Nature (2011)477:90-94; Villeda et al., “Young blood reverses age-related impairmentsin cognitive function and synaptic plasticity in mice.,” Nature medicine(2014) 20:659-663). Interestingly, heterochronic parabiosis studies haverevealed an age-dependent bi-directionality in the influence of thesystemic environment indicating pro-youthful factors in young bloodelicit rejuvenation while pro-aging factors in old blood drive aging(Katsimpardi et al., “Vascular and neurogenic rejuvenation of the agingmouse brain by young systemic factors.,” Science (2014) 344:630-634;Villeda et al., “The ageing systemic milieu negatively regulatesneurogenesis and cognitive function.,” Nature (2011) 477: 90-94; Ruckhet al., “Rejuvenation of regeneration in the aging central nervoussystem.,” Cell stem cell (2012) 10:96-103; Conboy et al., “Rejuvenationof aged progenitor cells by exposure to a young systemic environment.,”Nature (2005) 433:760-764; Brack et al., “Increased Wnt signaling duringaging alters muscle stem cell fate and increases fibrosis.,” Science(2007) 317: 807-810). It has been proposed that mitigating the effect ofpro-aging factors may also provide an effective approach to rejuvenateaging phenotypes (Villeda et al., “Young blood reverses age-relatedimpairments in cognitive function and synaptic plasticity in mice.,”Nature medicine (2014) 20:659-663; Laviano, “Young blood.,” The NewEngland journal of medicine (2014) 371:573-575; Bouchard & Villeda,“Aging and brain rejuvenation as systemic events.,” Journal ofneurochemistry (2014)).

In its traditional role, B2M represents the light chain of the MHC Imolecules that form an active part of the adaptive immune system(Zijlstra et al., “Beta 2-microglobulin deficient mice lack CD4-8+cytolytic T cells.,” Nature (1990) 344:742-746). In the central nervoussystem (CNS), B2M and MHC I can act independent of their canonicalimmune function to regulate normal brain development, synapticplasticity and even behavior (Lee et al. Synapse elimination andlearning rules co-regulated by MHC class I H2-Db. Nature (2014)509:195-200; Loconto et al., “Functional expression of murine V2Rpheromone receptors involves selective association with the M10 and M1families of MHC class Ib molecules.,” Cell (2003) 112:607-618; Boulanger& Shatz, “Immune signalling in neural development, synaptic plasticityand disease.,” Nature reviews. Neuroscience (2004) 5: 521-531; Shatz,“MHC class I: an unexpected role in neuronal plasticity.,” Neuron (2009)64:40-45; Huh et al., “Functional requirement for class I MHC in CNSdevelopment and plasticity.,” Science (2000) 290:2155-2159; Goddard etal., “Regulation of CNS synapses by neuronal MHC class I.,” Proceedingsof the National Academy of Sciences of the United States of America(2007) 104:6828-6833; Glynn et al., “MHCI negatively regulates synapsedensity during the establishment of cortical connections.,” Natureneuroscience (2011) 14:442-451). Additionally, in its soluble form, B2Maccumulates in the systemic blood circulation as a result of cellsurface shedding. Interestingly increased systemic levels of B2M havebeen implicated in cognitive impairments associated with chronichemodialysis (Murray, “Cognitive impairment in the aging dialysis andchronic kidney disease populations: an occult burden.,” Advances inchronic kidney disease (2008) 15:123-132; Corlin et al., “Quantificationof cleaved beta2-microglobulin in serum from patients undergoing chronichemodialysis.,” Clinical chemistry (2005) 51:1177-1184). Moreover,increase in soluble B2M has also been observed in the cerebral spinalfluid (CSF) of patients with HIV-dementia (McArthur et al., “Thediagnostic utility of elevation in cerebrospinal fluid beta2-microglobulin in HIV-1 dementia. Multicenter AIDS Cohort Study.,”Neurology (1992) 42:1707-1712; Brew et al., “Predictive markers of AIDSdementia complex: CD4 cell count and cerebrospinal fluid concentrationsof beta 2-microglobulin and neopterin.,” The Journal of infectiousdiseases (1996) 174:294-298) and Alzheimer's disease (Carrette et al.,“A panel of cerebrospinal fluid potential biomarkers for the diagnosisof Alzheimer's disease.” Proteomics (2003) 3:1486-1494), furtherimplicating B2M in cognitive dysfunction.

We first characterized changes in systemic levels of B2M in mouse plasmaduring normal aging (FIG. 1A,B), and in the experimental aging model ofheterochronic parabiosis (FIG. 1C,D). We observed a three-fold increasein B2M levels in plasma derived from aged compared to young animals(FIG. 1B), and detected a corresponding increase in B2M levels in plasmaderived from young heterochronic parabionts after exposure to aged bloodcompared to young isochronic parabionts (FIG. 1D). To corroboratesystemic changes observed for B2M in aging mice with systemic changesoccurring in humans, we measured B2M in archived plasma and CSF samplesfrom healthy individuals between 20 and 90 years of age (Table 1,above). We detected an age-related increase in B2M measured in bothplasma and CSF, consistent with changes observed in aging mice (FIG.1E,F). Having identified B2M as a potential pro-aging systemic factor wenext asked whether increasing B2M systemically could elicit cognitiveimpairments reminiscent of age-related dysfunction. As a control, wefirst tested hippocampal-dependent learning and memory using radial armwater maze (RAWM) and contextual fear conditioning paradigms in a cohortof young and old untreated animals and observed age-related cognitiveimpairments with both behavioral paradigms (FIG. 2A-E). Subsequently, wecognitively tested young adult mice systemically administered solubleB2M protein or vehicle through intraorbital injections (FIG. 1G).Animals showed no signs of illness or weight loss regardless oftreatment (FIG. 3A). During the training phase of the RAWM task all miceshowed similar swim speeds (FIG. 3B) and learning capacity for the task(FIG. 1H). However, during the testing phase animals receiving B2Mexhibited impaired learning and memory deficits, committingsignificantly more errors in locating the target platform than animalsreceiving vehicle control (FIG. 1H). During fear conditioning trainingall mice, regardless of treatment, exhibited no differences in baselinefreezing time (FIG. 3C). However, mice receiving B2M demonstrateddecreased freezing time during contextual (FIG. 1I), but not cued (FIG.3D), memory testing compared to vehicle treated control animals.Together, these behavioral data demonstrate that systemic administrationof exogenous B2M can impair learning and memory.

Impairments in hippocampal-dependent learning and memory have beenpreviously linked with decreased adult neurogenesis (Clelland et al., “Afunctional role for adult hippocampal neurogenesis in spatial patternseparation.,” Science (2009) 325:210-213; Kitamura et al., “Adultneurogenesis modulates the hippocampus-dependent period of associativefear memory.,” Cell (2009) 139:814-827; Zhang et al., “A role for adultTLX-positive neural stem cells in learning and behaviour.,” Nature(2008) 451:1004-1007). While a causal link between age-related cognitivedecline and decreased adult neurogenesis remains obfuscated (Drapeau etal., “Spatial memory performances of aged rats in the water maze predictlevels of hippocampal neurogenesis.,” Proceedings of the NationalAcademy of Sciences of the United States of America (2003)100:14385-14390: Merrill et al., “Hippocampal cell genesis does notcorrelate with spatial learning ability in aged rats.,” The Journal ofcomparative neurology (2003) 459:201-207; Bizon & Gallagher, “Productionof new cells in the rat dentate gyrus over the lifespan: relation tocognitive decline.,” The European journal of neuroscience (2003)18:215-219; Seib et al. “Loss of Dickkopf-1 restores neurogenesis in oldage and counteracts cognitive decline.” Cell stem cell (2013)12:204-214), recent studies using heterochronic parabiosis indicate thatcognitive changes elicited by blood are associated with correspondingchanges in adult neurogenesis (Katsimpardi et al., “Vascular andneurogenic rejuvenation of the aging mouse brain by young systemicfactors,” Science (2014) 344:630-634; Villeda et al., “The ageingsystemic milieu negatively regulates neurogenesis and cognitivefunction.,” Nature (2011) 477: 90-94). Consequently, we investigatedwhether decreased levels of adult hippocampal neurogenesis alsoaccompanied cognitive impairments elicited by increased systemicexposure to B2M. Using immunohistochemical analysis we detected asignificant decrease in the number of Doublecortin (Dcx)-positive newlyborn neurons (FIGS. 1J,K), and Mcm2-positive progenitors (FIG. 4A) inthe DG of mice systemically administrated exogenous B2M compared to miceinjected with vehicle control. Consistent with changes in neurogenesiswe detected a decrease in the number of proliferating cells havingincorporated Bromodeoxyuridne (BrdU) in animals injected with B2Mcompared to vehicle (FIG. 4B). These data indicate that systemicexposure to exogenous B2M is sufficient to decrease adult neurogenesis.

To determine whether systemic age-related changes in B2M levels werealso accompanied by local changes within the brain, we measured B2Mlevels within the hippocampus of young and aged animals by Western blotanalysis and detected an age-related increase in B2M protein (FIG. 5A).Subsequently, we asked whether systemic changes in the levels of B2M,elicited by heterochronic parabiosis, were also associated withcorresponding local changes within the young hippocampus after exposureto an old systemic environment. We detected an increase in B2M proteinexpression in the hippocampal lysates of young heterochronic parabiontscompared to young isochronic controls (FIG. 5B). Together, these datashow that age-related changes in B2M observed in the systemicenvironment are accompanied by corresponding changes within the brain.

To test the effect of local exposure to exogenous B2M on learning andmemory we administered B2M or vehicle control by bilateral stereotaxicinjections followed by cognitive testing using RAWM and contextual fearconditioning (FIG. 5C). All mice showed similar swim speeds (FIG. 6A)and learning capacity (FIG. 5D) during the training phase of the RAWM.During the testing phase animals receiving B2M committed significantlymore errors in locating the target platform than animals receivingvehicle control (FIG. 5D). During fear conditioning training no miceexhibited differences in baseline freezing time (FIG. 6B). However, micereceiving B2M demonstrated decreased freezing time during contextual(FIG. 5E), but not cued (FIG. 6C), memory testing. These functional dataindicate that local exposure to B2M in the DG impairs learning andmemory.

To examine the effect of local exposure to exogenous B2M in the brain,we stereotaxically injected B2M into the right DG and vehicle controlinto the left contralateral DG of young adult mice. Local exposure ofthe DG to B2M resulted in a decrease in the number of Dcx-positive cellscompared with the contralateral DG treated with vehicle control (FIG.5F,G). Given B2M is an active component of the MHC I complex throughnon-covalent interactions on the cell surface, we next investigatedwhether the inhibitory effect of exogenous B2M on adult neurogenesis wasmediated by MHC I cell surface expression. The transporter associatedwith antigen processing 1 (Tap1) protein is required for transport ofMHC I molecules to the cell surface, and absence of Tap1 results in veryfew classical MHC I molecules reaching the cell surface (Boulanger &Shatz, “Immune signalling in neural development, synaptic plasticity anddisease,” Nature reviews. Neuroscience (2004) 5:521-531; Shatz, “MHCclass I: an unexpected role in neuronal plasticity.,” Neuron (2009)64:40-45; Van Kaer at al., “TAP1 mutant mice are deficient in antigenpresentation, surface class I molecules, and CD4-8+ T cells.,” Cell(1992) 71:1205-1214). Therefore, to test whether decreased surface MHC Iexpression could mitigate the inhibitory effect of exogenous B2M, westereotaxically injected young adult Tap1 knock out mice (Tap1−/−) withB2M into the right DG and vehicle control into the left contralateralDG. No difference in the number of Dcx-positive cells was detectedbetween the B2M treated DG compared to the control treated DG of TAP1−/−mice (FIG. 5F,H). Consistent with previous reports (Laguna Goya et al.,“Adult neurogenesis is unaffected by a functional knock-out of MHC classI in mice,” Neuroreport (2010) 21:349-353), we observed no differencesin baseline levels of neurogenesis between young adult Tap1−/− and wildtype (WT) littermates (FIG. 7A). Together, these data suggest thatincreased local levels of exogenous B2M is sufficient to decrease adultneurogenesis in a classical MHC I dependent manner.

Next, we sought to investigate whether decreasing surface MHC Iexpression could also mitigate in part the negative effects of agedblood on adult neurogenesis elicited by heterochronic parabiosis (FIG.8A). Consistent with previous reports (Katsimpardi et al., “Vascular andneurogenic rejuvenation of the aging mouse brain by young systemicfactors.,” Science (2014) 344:630-634; Villeda et al., “The ageingsystemic milieu negatively regulates neurogenesis and cognitivefunction.,” Nature (2011) 477: 90-94), we observed a decrease in thenumber of Dcx-positive immature neurons (FIGS. 8B,C), Tbr2-positiveprogenitors (FIGS. 9A,B), and BrdU-positive proliferating cells (FIG.8D) in young wild type heterochronic compared to young wild typeisochronic parabionts. In contrast, we did not detect robust changes inthe levels of neurogenesis in young Tap1−/− heterochronic parabionts(FIGS. 8B-D). As a control, no changes in neurogenesis were detectedbetween young wild type and young Tap1−/− isochronic parabionts (FIGS.7B-D). Together, our data further substantiate the role of pro-agingfactors as drivers of regenerative impairment in the adult brain, andfurthermore implicate MHC I molecules in this process.

Lastly, we investigated the potential benefit abrogating endogenous B2Mexpression could have on the age-related cognitive decline observedduring aging. We utilized B2M knockout mice (B2M−/−), which lack proteinexpression both in the systemic environment and locally within thebrain. We assessed hippocampal-dependent learning and memory in youngand aged B2M−/− and WT controls using RAWM and contextual fearconditioning. In young animals no difference in spatial learning andmemory were observed between B2M−/− and WT controls during RAWM trainingor testing (FIG. 10A). Interestingly, aged B2M−/− mice showed enhancedspatial learning capacity during the training phase of the RAWMparadigm, as well as enhanced learning and memory for platform locationduring the testing phase of the task compared to WT controls (FIG. 10C).Animals in each age group showed no differences in swim speed regardlessof genotype (FIG. 11A,D). During fear conditioning training, all miceexhibited similar baseline freezing independent of genotype (FIG.11B,E). Additionally, no difference in freezing was observed in youngB2M−/− and WT mice during either contextual (FIG. 10B) or cued fearconditioning (FIG. 11C) paradigms. However, aged B2M−/− micedemonstrated significantly increased freezing in contextual (FIG. 10D),but not cued (FIG. 11F) memory testing compared to WT controls. Our dataindicate absence of endogenous B2M ameliorates age-related impairmentsin hippocampal-dependent learning and memory in old animals furtherimplicating B2M as a pro-aging factor mediating cognitive decline duringaging.

Subsequently, we investigated whether absence of endogenous B2M couldalso counteract age-related decline in adult neurogenesis. We examinedchanges in neurogenesis in young and aged adult B2M−/− and WTlittermates by immunohistochemical analysis. We observed that in youngadult animals the absence of endogenous B2M expression had no effect onthe number of Dcx-positive cells (FIG. 10E,F). Importantly, in agedanimals we observed an almost 2-fold increase in the number ofDcx-positive newly born neurons in B2M−/− mice compared to wild typelittermates (FIG. 10E,G). Consistent with our Dcx data, we did notdetect changes in proliferation by BrdU labeling in young animalsregardless of B2M expression (FIG. 12A). However, aged B2M−/− animalsshowed a significant increase in the number of BrdU-positive cellscompared to WT controls (FIG. 12B). Subsequently, we assessed neuronaldifferentiation and survival in B2M−/− mice using a long-term BrdUincorporation paradigm, in which mature differentiated neurons expressboth BrdU and the neuronal marker NeuN (FIG. 10H). Young adult B2M−/−mice showed no differences in the percentage of cells expressing bothBrdU and NeuN compared to their WT counterparts (FIG. 10I); while agedadult B2M−/− mice showed a significant increase in the percentage ofcells expressing both BrdU and NeuN compared to age matched WTlittermates (FIG. 10J). We detected no differences in astrocytedifferentiation at any age quantified as the percentage of cellsexpressing BrdU and GFAP markers (FIG. 12C-E). Together, these dataindicate that loss of endogenous B2M enhances the levels of neurogenesisin the aged brain, but not the young brain, indicating an active rolefor B2M in the decline in regenerative capacity during aging.

B2M in concert with MHC I molecules continue to be demonstrated to havea unique involvement in the CNS (Lee et al., “Synapse elimination andlearning rules co-regulated by MHC class I H2-Db.,” Nature (2014)509:195-200; Boulanger & Shatz, “Immune signalling in neuraldevelopment, synaptic plasticity and disease.,” Nature reviews.Neuroscience (2004) 5:521-531; Shatz, “MHC class I: an unexpected rolein neuronal plasticity.,” Neuron (2009) 64:40-45; Huh et al.,“Functional requirement for class I MHC in CNS development andplasticity.,” Science (2000) 290:2155-2159; Goddard et al., “Regulationof CNS synapses by neuronal MHC class I.,” Proceedings of the NationalAcademy of Sciences of the United States of America (2007)104:6828-6833; Glynn et al., “MHCI negatively regulates synapse densityduring the establishment of cortical connections.,” Nature neuroscience(2011) 14:442-451), with recent studies beginning to explore thecurative effect of abrogating MHC I expression in brain injury modelssuch as stroke (Adelson et al., “Neuroprotection from stroke in theabsence of MHCI or PirB.” Neuron (2012) 73:1100-1107). However, thefunctional involvement of these molecules during aging in either the CNSor peripheral tissues had not yet been investigated. Our study thuselucidates a previously unrecognized role for B2M in the progression ofage-related impairments in both cognitive and regenerative processes.Moreover, our study also implicates MHC I surface expression inmediating, in part, the negative effects of B2M and heterochronicparabiosis on regenerative function. Notably, human Genome-WideAssociation studies (GWAS) have linked the MHC locus on chromosome 6p21with degenerative diseases of aging, suggesting an active role for thesemolecules in age-dependent impairments (Jeck et al., “Review: ameta-analysis of GWAS and age-associated diseases.,” Aging cell (2012)11:727-731). Our data now provide functional evidence for such aninvolvement in aging phenotypes. Our data provide mechanistic insightinto how aging-related changes in the systemic environment driveimpairments locally in the aged brain, and highlight the involvement ofB2M and MHC I molecules in this process. From a translationalperspective, our data show that age-related cognitive and regenerativedysfunction observed during aging could be ameliorated by targeting B2Mat old age.

III. Determination of Relative Levels of B2M

Relative levels of beta2-microglobulin were determined in plasma samplesof healthy male human donors of 18, 30, 45, 55, and 66 years of age bythe SomaScan Proteomic Assay (Somalogic, Inc, Boulder, Colo.). For eachage group, plasma from 40 individuals was analyzed as 8 pools of 5individuals per pool. Statistical analysis was performed by two-sidedStudent's t-test of log-transformed values, and also by trend-analysisof untransformed data using the Jonckheere-Terpstra test. Observedchanges were found to be highly significant with the p-value of thet-test being 1.1×10⁻⁴ (66 vs 18 year old) and the p-value for theJT-test being 1.3×10⁻⁷ (all age groups). (RFU refers to “relativefluorescence units” by SomaScan Proteomic Assay.) The results aregraphically illustrated in FIG. 13.

Notwithstanding the appended clauses, the disclosure is also defined bythe following clauses:

1. A method of treating an adult mammal for an aging-associatedimpairment, the method comprising:

-   -   reducing the β2-microglobulin (B2M) level in the mammal in a        manner sufficient to treat the adult mammal for the        aging-associated impairment.        2. The method according to Clause 1, wherein the method        comprises reducing systemic B2M of the mammal.        3. The method according to Clause 2, wherein systemic B2M of the        mammal is reduced by removing B2M from blood of the mammal.        4. The method according to Clause 3, wherein the method        comprises extra-corporally removing B2M from blood of the        mammal.        5. The method according to Clause 1, wherein the B2M level is        reduced by administering to the mammal an effective amount of a        B2M level reducing agent.        6. The method according to Clause 5, wherein the B2M level        reducing agent is a B2M binding agent.        7. The method according to Clause 6, wherein the B2M binding        agent comprises an antibody or binding fragment thereof.        8. The method according to Clause 6, wherein the B2M binding        agent comprises a small molecule.        9. The method according to Clause 5, wherein the B2M level        reducing agent comprises a B2M expression inhibitor agent.        10. The method according to Clause 9, wherein the B2M expression        inhibitory agent comprises a nucleic acid.        11. The method according to any of the preceding clauses,        wherein the mammal is a primate.        12. The method according to Clause 11, wherein the primate is a        human.        13. The method according to any of the preceding clauses,        wherein the adult mammal is an elderly mammal.        14. The method according to Clause 13, wherein the elderly        mammal is a human that is 60 years or older.        15. The method according to any of the preceding clauses,        wherein the aging-associated impairment comprises a cognitive        impairment.        16. The method according to any of the preceding clauses,        wherein the adult mammal suffers from an aging associated        disease condition.        17. The method according to any of the preceding clauses,        wherein the aging associated disease condition is a cognitive        decline disease condition.

The preceding merely illustrates the principles of the invention. Itwill be appreciated that those skilled in the art will be able to devisevarious arrangements which, although not explicitly described or shownherein, embody the principles of the invention and are included withinits spirit and scope. Furthermore, all examples and conditional languagerecited herein are principally intended to aid the reader inunderstanding the principles of the invention and the conceptscontributed by the inventors to furthering the art, and are to beconstrued as being without limitation to such specifically recitedexamples and conditions. Moreover, all statements herein recitingprinciples, aspects, and embodiments of the invention as well asspecific examples thereof, are intended to encompass both structural andfunctional equivalents thereof. Additionally, it is intended that suchequivalents include both currently known equivalents and equivalentsdeveloped in the future, i.e., any elements developed that perform thesame function, regardless of structure. The scope of the presentinvention, therefore, is not intended to be limited to the exemplaryembodiments shown and described herein. Rather, the scope and spirit ofthe present invention is embodied by the appended claims.

1. A method of treating an adult mammal for an aging-associatedimpairment, the method comprising: reducing the β2-microglobulin (B2M)level in the mammal in a manner sufficient to treat the adult mammal forthe aging-associated impairment.
 2. The method according to claim 1,wherein the method comprises reducing systemic B2M of the mammal. 3.(canceled)
 4. (canceled)
 5. The method according to claim 1, wherein theB2M level is reduced by administering to the mammal an effective amountof a B2M level reducing agent.
 6. The method according to claim 5,wherein the B2M level reducing agent is a B2M binding agent.
 7. Themethod according to claim 6, wherein the B2M binding agent comprises anantibody or binding fragment thereof.
 8. The method according to claim6, wherein the B2M binding agent comprises a small molecule.
 9. Themethod according to claim 5, wherein the B2M level reducing agentcomprises a B2M expression inhibitor agent.
 10. The method according toclaim 9, wherein the B2M expression inhibitory agent comprises a nucleicacid.
 11. The method according to claim 1, wherein the mammal is aprimate.
 12. The method according to claim 11, wherein the primate is ahuman.
 13. The method according to claim 1, wherein the adult mammal isan elderly mammal.
 14. The method according to claim 13, wherein theelderly mammal is a human that is 60 years or older.
 15. The methodaccording to claim 1, wherein the aging-associated impairment comprisesa cognitive impairment.